U.S. patent number 10,570,370 [Application Number 15/506,736] was granted by the patent office on 2020-02-25 for method for differentiation into retinal ganglion cells from stem cells.
This patent grant is currently assigned to Seoul National University Hospital, Seoul National University R&DB Foundation. The grantee listed for this patent is SEOUL NATIONAL UNIVERSITY HOSPITAL, SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION. Invention is credited to Ji Yeon Kim, Sung Sup Park.
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United States Patent |
10,570,370 |
Park , et al. |
February 25, 2020 |
Method for differentiation into retinal ganglion cells from stem
cells
Abstract
Provided are a method of preparing retinal ganglion cells by
differentiation of stem into retinal ganglion cells, retinal
ganglion cells differentiated by the method, a method of screening
for a death inhibitor or a proliferation promoter of retinal
ganglion cells using the retinal ganglion cells differentiated by
the method, a kit of screening for the death inhibitor or the
proliferation promoter of retinal ganglion cells including the
retinal ganglion cells differentiated by the method, a
pharmaceutical composition for treating glaucoma or optic
neuropathy including the retinal ganglion cells, a method of
treating glaucoma or optic neuropathy including the step of
administering the retinal ganglion cells to a subject suspected of
having glaucoma or optic neuropathy, and a method of preparing a
mature retinal ganglion cell line.
Inventors: |
Park; Sung Sup (Seoul,
KR), Kim; Ji Yeon (Seoul, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION
SEOUL NATIONAL UNIVERSITY HOSPITAL |
Seoul
Seoul |
N/A
N/A |
KR
KR |
|
|
Assignee: |
Seoul National University R&DB
Foundation (Seoul, KR)
Seoul National University Hospital (Seoul,
KR)
|
Family
ID: |
55400066 |
Appl.
No.: |
15/506,736 |
Filed: |
August 27, 2015 |
PCT
Filed: |
August 27, 2015 |
PCT No.: |
PCT/KR2015/009004 |
371(c)(1),(2),(4) Date: |
February 25, 2017 |
PCT
Pub. No.: |
WO2016/032263 |
PCT
Pub. Date: |
March 03, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180016552 A1 |
Jan 18, 2018 |
|
Foreign Application Priority Data
|
|
|
|
|
Aug 27, 2014 [KR] |
|
|
10-2014-0112638 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
27/00 (20180101); A61K 35/30 (20130101); A61P
27/06 (20180101); A61P 27/02 (20180101); C12N
5/0621 (20130101); G01N 33/5058 (20130101); C12N
2501/599 (20130101); C12N 2506/02 (20130101); C12N
2506/45 (20130101); C12N 2501/41 (20130101); C12N
2501/415 (20130101); C12N 2501/105 (20130101); C12N
2501/155 (20130101); C12N 2501/115 (20130101); C12N
2501/385 (20130101); G01N 2800/168 (20130101) |
Current International
Class: |
C12N
5/079 (20100101); A61K 35/30 (20150101); G01N
33/50 (20060101) |
Field of
Search: |
;424/93.7 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
10-1268741 |
|
Apr 2011 |
|
KR |
|
2011043591 |
|
Apr 2011 |
|
WO |
|
2011043592 |
|
Apr 2011 |
|
WO |
|
Other References
Chen et al., "Generation of retinal ganglion-like cells from
reprogrammed mouse fibroblasts" IOVS Nov. 2010, vol. 51, No. 11, p.
5970-5978. cited by applicant .
Japanese Office Action for 2017-531436 dated Feb. 27, 2018. cited
by applicant .
Loubradou-Bourges, Supplementary European Search Report for EP
15836684, dated Feb. 15, 2018. cited by applicant .
Jagatha et al., "In vitro differentiation of retinal ganglion-like
cells from embryonic stem cell derived neural progenitors"
Biochemical and Biophysical Research Communications, 2009, vol.
380, p. 230-235. cited by applicant .
Tucker et al., "Use of a synthetic xeno-free culture substrate for
induced pluripotent stem cell induction and retinal
differentiation" Stem Cells Translational Medicine, 2013, vol. 2,
p. 16-24. cited by applicant .
Gill et al., "Methods of retinal ganglion cell differentiation from
pluripotent stem cells" Transnational Vision Science and
Technology, 2014, vol. 3, No. 4, p. 1-13. cited by applicant .
Barres, Barbara, A., et al., "Immunological, Morphological, and
Electrophysiological Variation among Retinal Ganglion Cells
Purified by Panning", Neron, vol. 1, (Nov. 1988), pp. 791-803.
cited by applicant .
Bull, Natalie, D., et al., "Use of Adult Rat Retinal Explant Model
for Screening of Potential Retinal Ganglion Cell Neuroprotective
Therapies", Investigative Opthalmology & Visual Science, vol.
52, No. 6, (May 2011), pp. 3309-3320. cited by applicant .
Connors, B.W., et al., "Electrophysiological Properties of
Neocortical Neurons in Vitro", Journal of Neurophysiology, vol. 48,
No. 6, (Dec. 1982), pp. 1302-1320. cited by applicant .
Jagatha, B., et al., "In vitro differentiation of retinal
ganglion-like cells from embryonic stem cell derived neural
progenitors", Biochem Biophys Res Commun, vol. 380, No. 2, Mar. 6,
2009, pp. 230-235. cited by applicant .
McCormick, David, A., et al., "Post-Natal Development of
Electrophysiological Properties of Rat Cerebral Cortical Pyramidal
Neurones", J. Physiol., vol. 393, (1987), pp. 743-762. cited by
applicant .
Osakada, Fumitaka, et al., "Stepwise differentiation of pluripotent
stem cells into retinal cells", Nature Protocols, vol. 4, No. 6,
(2009), pp. 811-824. cited by applicant .
Parameswaran, Sowmya, et al., "Induced Pluripotent Stem Cells
Generate Both Retinal Ganglion Cells and Photoreceptors:
Therapeutic Implications in Degenerative Changes in Glaucoma and
Age-Related Macular Degeneration", Stem Cells, vol. 28, No. 4,
(Apr. 2010), pp. 695-703. cited by applicant .
Zuber, Michael, E., et al., "Specification of the vertebrate eye by
a network of eye field transcription factors", Development, vol.
130, No. 21, (Nov. 2003), pp. 5155-5167. cited by
applicant.
|
Primary Examiner: Tichy; Jennifer M. H.
Attorney, Agent or Firm: Greer, Burns, and Crain, Ltd.
Einhorn; Gregory P.
Claims
The invention claimed is:
1. A method of preparing mature retinal ganglion cells by
differentiation of retinal progenitor cells into mature retinal
ganglion cells, comprising: (a) culturing the retinal progenitor
cells in a first medium comprising an IGF1R (insulin-like growth
factor-1receptor) activator and a Wnt signaling pathway activator
to differentiate the retinal progenitor cells into immature retinal
ganglion cells; and (b)(i) removing the Wnt signaling pathway
activator from the first medium to generate a second medium lacking
the Wnt signaling pathway activator, and further culturing the
differentiated immature retinal ganglion cells of step (a) in the
second medium lacking the Wnt signaling pathway activator, wherein
the further culturing induces differentiation of the immature
retinal ganglion cells into mature retinal ganglion cells, or
(b)(ii) removing the differentiated immature retinal ganglion cells
of step (a) to a second medium whose composition does not comprise
Wnt signaling pathway activator, and culturing the differentiated
immature retinal ganglion cells in the second medium, wherein the
culturing induces differentiation of the immature retinal ganglion
cells into mature retinal ganglion cells, wherein the
differentiated mature retinal ganglion cells of (b)(i) or (b)(ii)
express one or more genes selected from the group consisting of
Thy1.2, TrkB, NMDAR1, Map2, Vglut1, PSD-95, Synaptophysin, and
Synapsin1.
2. The method of claim 1, wherein the first medium further
comprises: a BMP (bone morphogenetic protein) signaling pathway
inhibitor, or an FGF (fibroblast growth factor) signaling pathway
activator; or a BMP (bone morphogenetic protein) signaling pathway
inhibitor and an FGF (fibroblast growth factor) signaling pathway
activator.
3. The method of claim 2, wherein the second medium of (b)(i) or
(b)(ii) does not comprise a BMP signaling pathway inhibitor and a
FGF signaling pathway activator; and does comprise a Shh (sonic
hedgehog) signaling pathway activator.
4. The method of claim 3, further comprising: (c)(i) culturing the
mature retinal ganglion cells in a third medium comprising RA
(retinoic acid); or (c)(ii) adding a retinoic acid (RA) to the
second medium of (b)(i) or (b)(ii) to generate a third medium and
culturing the mature retinal ganglion cells in the third
medium.
5. The method of claim 4, further comprising: (d) removing the
IGF1R activator, the Shh signaling pathway activator or the
retinoic acid (RA) from the third medium and culturing the mature
retinal ganglion cells.
6. The method of claim 1, wherein the retinal progenitor cells of
(a) are obtained by a method comprising: (1) culturing stem cells
in a medium comprising an IGF1R activator, a Wnt signaling pathway
inhibitor, and a BMP signaling pathway inhibitor to differentiate
the stem cells into eye field precursors in the form of floating
aggregates; and (2) culturing the eye field precursors in the form
of floating aggregates of (1) in a medium whose composition
comprises an FGF signaling pathway activator to differentiate the
eye field precursors in the form of floating aggregates into
retinal progenitor cells.
7. The method of claim 6, wherein (1) is performed for about 1 day
to 30 days, and (2) is performed for about 5 days to 15 days.
8. The method of claim 6, wherein the stem cells are selected from
the group consisting of bone marrow stem cells (BMS), cord blood
stem cells, amniotic fluid stem cells, fat stem cells, retinal stem
cells (RSCs), intraretinal Muller glial cells, embryonic stem cells
(ESCs), induced pluripotent stem cells (iPSCs), and somatic cell
nuclear transfer cells (SCNTCs).
9. The method of claim 1, wherein (a) is performed for about 1 day
to 10 days, and (b)(i) or (b)(ii) is performed for about 1 day to
120 days.
10. The method of claim 1, wherein the IGF1R activator is IGF-1 or
IGF-2.
11. The method of claim 1, wherein a concentration of IGF1R
activator in the first medium and the second medium of (b)(i) or
(b)(ii) is about 0.01 ng/mL to about 100 ng/mL.
12. The method of claim 1, wherein the Wnt signaling pathway
activator is one or more selected from the group consisting of
Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a,
Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and
Wnt16b, substance increasing p3-catenin levels; lithium, LiCl,
bivalent zinc, BIO (6-bromoindirubin-3'-oxime), SB216763, SB415286,
CHIR99021, QS11 hydrate, TWS119, Kenpaullone, alsterpaullone,
indirubin-3'-oxime, TDZD-8 and Ro 318220 methanesulfonate salt;
Axin inhibitors; APC (adenomatous polyposis coli) inhibitors;
norrin and R-spondin 2.
13. The method of claim 1, wherein the Wnt signaling pathway
activator is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b,
Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b,
Wnt11, or Wnt16b, and is present in the first medium at a
concentration of about 0.01ng/mL to about 500ng/mL; the Wnt
signaling pathway activator is LiCl and is present in the first
medium at a concentration of about 0.1mM to 50mM; the Wnt signaling
pathway activator is BIO (6-bromoindirubin-3'-oxime),and is present
in the first medium at a concentration of about 0.1.mu.M to
50.mu.M; or the Wnt signaling pathway activator is SB415286 , and
is present in the first medium at a concentration of about 0.1.mu.M
to 500.mu.M.
14. The method of claim 2, wherein the BMP signaling pathway
inhibitor is one or more selected from the group consisting of
Noggin, chordin, twisted gastrulation (Tsg), cerberus, coco,
gremlin, PRDC (protein related to DAN and Cerberus), DAN
(differential screening-selected gene aberrative in neuroblastoma),
dante, follistatin, USAG-1(uterine sensitization-associated gene
1), dorsomorphin and sclerostin.
15. The method of claim 2, wherein a concentration of BMP signaling
pathway inhibitor in the first medium is about 0.01ng/mL to about
10ng/mL.
16. The method of claim 2, wherein the FGF signaling pathway
activator is one or more selected from the group consisting of
FGF1, FGF2, FGF4, FGF8, FGF9, FGF17 and FGF19.
17. The method of claim 2, wherein a concentration of FGF signaling
pathway activator in the first medium is about 0.01ng/mL to about
100ng/mL.
18. The method of claim 3, wherein the Shh signaling pathway
activator is one or more selected from the group consisting of Shh,
Smo (smoothened) receptor activators, inhibitors of Smo inhibition
by Ptc (Patched), substances increasing Ci/Gli family levels,
inhibitors of the intracellular degradation of Ci/Gli proteins, and
Shh receptor activator Hg-Ag and purmorphamine.
19. The method of claim 3, wherein a concentration of the Shh
signaling pathway activator in the second medium is about 0.1ng/mL
to about 5000ng/mL.
20. The method of claim 4, wherein a concentration of RA in the
third medium of (c)(i) or (c)(ii) is about 0.5nM to about
10,000nM.
21. The method of claim 6, wherein the Wnt signaling pathway
inhibitor is one or more selected from the group consisting of
Dkk-1, Dkk-2, Dkk-3 and Dkk-4.
22. The method of claim 6, wherein a concentration of the Wnt
signaling pathway inhibitor in the medium of (1) is about 0.01ng/mL
to about 10,000ng/mL.
23. The method of claim 1, further comprising the step of
determining whether the retinal progenitor cells are differentiated
into the mature retinal ganglion cells.
24. The method of claim 23, wherein the step of determining whether
the retinal progenitor cells are differentiated into the mature
retinal ganglion cells is performed by measuring mRNA or protein
expression levels of one or more genes selected from the group
consisting of Thy1.2, TrkB, NMDAR1, Map2, Vglut1, PSD-95,
Synaptophysin, and Synapsin1.
25. The method of claim 6, wherein the eye field precursors in the
form of floating aggregates are cultured in (2) by adhering to a
plate coated with an extracellular matrix selected from the group
consisting of poly-D-lysine, laminin, poly-L-lysine, matrigel,
agar, polyornithine, gelatin, collagen, fibronectin and
vitronectin.
26. The method of claim 6, wherein the medium of (1) further
comprises DMEM/F12, 10% KnockOut serum replacement, and 1% B27
supplement; and the medium of (2) further comprises DMEM/F12, about
1% B27 supplement, and about 1% N2 supplement.
27. The method of claim 1, wherein the first medium further
comprises DMEM/F12, about 1% B27 supplement, and about 1% N2
supplement.
28. The method of claim 1, wherein the immature retinal ganglion
cells express: one or more genes selected from the group consisting
of Brn3B, Brn3A, Islet-1, NF200 and Tuj1; and Math5.
29. The method of claim 1, wherein the mature retinal ganglion
cells are present at from about 60% to about 95% or more of total
cells after the culturing of (b)(i) or (b)(ii).
30. The method of claim 6, wherein the BMP signaling pathway
inhibitor is one or more selected from the group consisting of
Noggin, chordin, twisted gastrulation (Tsg), cerberus, coco,
gremlin, PRDC (protein related to DAN and Cerberus), DAN
(differential screening-selected gene aberrative in neuroblastoma),
dante, follistatin, USAG-1(uterine sensitization-associated gene
1), dorsomorphin and sclerostin.
31. The method of claim 6, wherein a concentration of the BMP
signaling pathway inhibitor in the medium of (1) is 0.01ng/mL to
100ng/mL.
32. The method of claim 6, wherein the FGF signaling pathway
activator is one or more selected from the group consisting of
FGF1,FGF2, FGF4, FGF8, FGF9, FGF17 and FGF19.
33. The method of claim 6, wherein a concentration of the FGF
signaling pathway activator in the medium of (2) is 0.01ng/mL to
100ng/mL.
Description
RELATED APPLICATIONS
This application is a national phase application claiming benefit
of priority under 35 U.S.C. .sctn. 371 to International (PCT)
Patent Application serial number PCT/KR2015/009004, filed Aug. 27,
2015, which claims benefit of priority to Korean Application
10-2014-0112638, filed Aug. 27, 2014. The aforementioned
applications are expressly incorporated herein by reference in
their entirety and for all purposes.
TECHNICAL FIELD
The present invention relates to a method of preparing retinal
ganglion cells by differentiation of stem cells into retinal
ganglion cells, retinal ganglion cells differentiated by the
method, a method of screening for a death inhibitor or a
proliferation promoter of retinal ganglion cells using the retinal
ganglion cells differentiated by the method, a kit of screening for
the death inhibitor or the proliferation promoter of retinal
ganglion cells including the retinal ganglion cells differentiated
by the method, a pharmaceutical composition for treating glaucoma
or optic neuropathy including the retinal ganglion cells, a method
of treating glaucoma or optic neuropathy including the step of
administering the retinal ganglion cells to a subject suspected of
having glaucoma or optic neuropathy, and a method of preparing a
mature retinal ganglion cell line.
BACKGROUND
Blindness is the medical condition of lacking visual perception. As
many as tens of millions of people, which account for 0.2% to 0.5%
of the population of the world, are affected by blindness, and
suffer from great losses in personal, social and economical
respects. Retinal glaucoma or optic neuropathy is one of the
leading causes of blindness worldwide. Glaucoma is the most common
progressive optic neuropathy, leading to irreversible blindness.
The global prevalence of glaucoma for people over 40 is as high as
about 2% to 3%. Globally, there were an estimated 60 million people
with glaucoma in 2010. The prevalence of glaucoma is expected to
increase to 80 million by 2020 (Quigley H A and Broman A T, Br J
Ophthalmol 2006; 90: 262-267). The prevalence of glaucoma in the
socially active middle-aged generation is 2% to 3%, and it is
expected that the number of patients will greatly increase as the
population ages, which will become a social and economic burden.
Currently, reduction of intraocular pressure is the only method
that is clinically applied for glaucoma treatment, but it is known
that a significant proportion of glaucoma patients still progress
to blindness. It is known that reduction of intraocular pressure is
a conservative method capable of merely inhibiting glaucoma
progression, and treatment of the underlying glaucoma is
impossible.
Meanwhile, optic neuropathy generally describes optic nerve
abnormalities caused by different factors, and includes optic
neuritis, ischemic optic neuropathy, toxic or deficiency optic
neuropathy, hereditary optic neuropathy, optic atrophy, etc. Among
them, the diseases caused by optic nerve degeneration and damage
can be helped by stem cell therapy.
Specifically, glaucoma is caused by degeneration and loss of
retinal ganglion cells (RGCs). After passing through the eye, light
is converted to electric signals in photoreceptor cells, and
retinal ganglion cells transmit these electric signals to the
central optic nerve of the brain.
On the other hand, stem cell/regenerative therapy may be the best
therapy for glaucoma and optic neuropathy. A disease caused by
degeneration and loss of a single cell type is a strong target of
stem cell therapy. In particular, because the eye is very easily
accessible for surgical manipulation, and various surgical
procedures have been established, there are no difficulties in the
application of stem cells to lesions, and the eye can be a model
for the development of therapeutic drugs.
The goals of stem cell therapy in glaucoma and optic neuropathy are
1) to replace degenerative and damaged RGCs with new RGCs (cell
replacement therapy, neuro-regeneration), 2) to induce therapeutic
effects of anti-inflammation, anti-cell death, neuroprotection, and
vascular protection on degenerative and damaged RGCs as paracrine
effects of stem cell therapy, and 3) to select and develop new
drugs using stem cells for the treatment of glaucoma, for which
direct therapeutic agents have not yet been developed. This therapy
may lead to a personalized stem cell therapy using patient-derived
pluripotent stem cells. Highly efficient production of retinal
ganglion cells is essential in the development of therapeutic
compositions or models for therapeutic drugs. In particular, there
is a problem in that retinal ganglion cells degenerated or damaged
by diseases cannot be prepared in vitro, and therefore, it is
impossible to develop new drugs for glaucoma and optic neuropathy
using normal or abnormal retinal ganglion cells. Therefore, if a
large amount of retinal ganglion cells are produced from stem
cells, it is possible to create disease models for retinal ganglion
cell-associated diseases such as glaucoma and optic neuropathy, and
therefore, drugs for the diseases can be easily developed.
Especially, production of RGCs differentiated from patient-derived
induced pluripotent stem cells (iPSC-RGCs) makes it possible to
develop new drugs capable of preventing and inhibiting optic nerve
degeneration. In addition, the present inventors disclosed a method
of differentiating retinal cells from stem cells in the previous
patents (Korean Patent NO. 10-1268741 (2013 May 22) and
WO2011/043591 (2011 Apr. 14)). However, according to the methods
disclosed in the above documents, only about 6% of retinal
progenitor cells are differentiated into retinal ganglion cells.
Accordingly, there is still a need to develop a differentiation
method capable of maximizing differentiation into retinal ganglion
cells.
DISCLOSURE
Technical Problem
The present inventors have tried to produce retinal ganglion cells
which are differentiated from human embryonic stem cells, but they
showed poor differentiation. Accordingly, the present inventors
have made many efforts to develop a new method capable of producing
retinal ganglion cells from stem cells. As a result, they developed
a method of differentiating human embryonic stem cells into retinal
ganglion cells with a high yield for a short period of time of 5
weeks under chemically defined culture conditions without gene
implantation or co-culture with retinal tissues. They found that
the retinal ganglion cells developed according to the method of the
present invention are about 200-fold higher in population than the
starting human embryonic stem cells, and the retinal ganglion cells
exhibit excellent neurophysiological functions, thereby completing
the present invention.
Technical Solution
An object of the present invention is to provide a method of
preparing mature retinal ganglion cells by differentiation of stem
cells into mature retinal ganglion cells, comprising (a) culturing
retinal progenitor cells in a medium containing an IGF1R
(insulin-like growth factor-1 receptor) activator and a Wnt
signaling pathway activator to differentiate them into immature
retinal ganglion cells; and (b) culturing the immature retinal
ganglion cells in a medium prepared by removing the Wnt signaling
pathway activator from the medium of step (a) and adding the IGF1R
activator thereto.
Another object of the present invention is to provide a method of
preparing mature retinal ganglion cells by differentiation of
immature retinal ganglion cells into mature retinal ganglion cells,
including the step of culturing immature retinal ganglion cells in
a medium containing an IGF1R activator and an Shh (sonic hedgehog)
signaling pathway activator.
Still another object of the present invention is to provide a
method of screening for a death inhibitor or a proliferation
promoter of mature retinal ganglion cells, comprising (a) treating
the mature retinal ganglion cells obtained and isolated by the
above method with a death inhibitor candidate or a proliferation
promoter candidate; and (b) determining that the candidate is a
death inhibitor or a proliferation promoter of mature retinal
ganglion cells when the candidate inhibits death of the mature
retinal ganglion cells or promotes proliferation of the mature
retinal ganglion cells, compared to a non-candidate-treated
group.
Still another object of the present invention is to provide
immature retinal ganglion cells and mature retinal ganglion cells
prepared according to the above-described method of the present
invention.
Still another object of the present invention is to provide a kit
of screening for a death inhibitor or a proliferation promoter of
mature retinal ganglion cells, including the mature retinal
ganglion cells.
Still another object of the present invention is to provide a
pharmaceutical composition for treating glaucoma or optic
neuropathy, including the mature retinal ganglion cells.
Still another object of the present invention is to provide a
method of treating glaucoma or optic neuropathy including the step
of administering the mature retinal ganglion cells to a subject
suspected of having glaucoma or optic neuropathy.
Still another object of the present invention is to provide a
method of preparing a mature retinal ganglion cell line, comprising
(a) culturing retinal progenitor cells in a medium containing an
IGF1R activator and a Wnt signaling pathway activator to
differentiate them into immature retinal ganglion cells; and (b)
culturing the immature retinal ganglion cells in a medium prepared
by removing the Wnt signaling pathway activator from the medium of
step (a) and adding the IGF1R activator thereto.
Advantageous Effects
According to the method of the present invention, retinal ganglion
cells can be differentiated from stem cells with a high yield, and
the retinal ganglion cells obtained by the method can be used in a
selection test of therapeutic agents for diseases such as glaucoma
or optic neuropathy and also used in cell therapy.
DESCRIPTION OF DRAWINGS
FIG. 1A-1B shows cytomorphological microphotographs.
FIG. 1A shows cell floc of human embryonic stem cells in an
undifferentiated state (30 passages): after being cultured for 5
days from cells of passage number 29. The cells are typical cell
floc of human embryonic stem cells in an undifferentiated state,
characterized by definite separation from adjacent mouse embryonic
fibroblast feeder cells and having a plain surface and uniform
morphology.
FIG. 1B shows floating aggregates, in which the floating aggregates
were cultured for 4 days in ultra-low attachment plates after being
isolated from the undifferentiated human embryonic stem cell floc
of FIG. 1(A). The floating aggregates have spherical morphology,
and one floating aggregate consists of about 292.+-.53 cells.
FIG. 1C-1F shows cytomorphological microphotographs of cells
differentiating into retinal ganglion cells.
FIG. 1C shows cells on day 14 after induction of the
differentiation: Cell morphology after transferring the floating
aggregates to poly-D-lysine/laminin-coated plates and culturing
them for 10 days therein, that is, on day 14 after induction of the
differentiation of the undifferentiated human embryonic stem cells.
It was observed that the cells were separated from the floating
aggregates and underwent differentiation, and had a morphological
character of the early stage of differentiation, with meager
cytoplasm and round, large nuclei.
FIG. 1D shows cells on day 17 after induction of the
differentiation: Cell morphology on day 17 after induction of the
differentiation of the undifferentiated human embryonic stem cells.
The cells differentiated with concomitant active proliferation, and
cell flocs under active proliferation and differentiation formed a
round flower-shaped rosette configuration.
FIG. 1E shows cells on day 22 after induction of the
differentiation: The cells became richer in cytoplasm and their
nuclei were smaller than those of FIG. 1(D) as the differentiation
progressed. It was observed that cell flocs differentiated into
neurons with short or long neuronal axons.
FIG. 1F shows cells on day 39 after induction of the
differentiation: Most cells differentiated into neurons. With the
progress of differentiation into neurons, the cells showed the same
cellularity, but had a mature neuronal shape, compared to those on
day 22 after induction of the differentiation in FIG. 1(E). The
nerve cell bodies formed a plurality of clusters, which were
connected with each other by long neuronal axons.
*Microscopic field: FIG. 1A-1F (left: 40.times. magnification;
right: 100.times. magnification).
FIG. 2A-D shows characteristics of induced pluripotent stem cells
reprogrammed from human neonatal foreskin BJ-1 fibroblasts.
FIG. 2A shows phase-contrast microscopic images of human induced
pluripotent stem cells: Cell line after being cultured for 6 days
from cells of passage number 6. The stem cells are characterized by
definite separation from adjacent mouse embryonic body cells as
feeder cells and having a plain surface and uniform morphology.
FIGS. 2B and 2C are alkaline phosphatase, Nanog, and SSEA-4
staining results showing the characteristics of human embryonic
stem cells of induced pluripotent stem cells.
FIG. 2D is the result of teratoma assay for evaluating pluripotency
of induced pluripotent stem cells generated from fibroblasts. At 10
weeks after transplantation of induced pluripotent stem cells
generated from fibroblasts into the dorsal surface of
immunosuppressed SCID mouse, induced pluripotent stem cells
developed teratomas with three germ layers of ectoderm (left:
neural tissues), mesoderm (middle: cartilage), and endoderm (right:
digestive system), indicating pluripotency.
*Scale bar: FIG. 2A-2C: 200 .mu.m.
FIG. 3 is a schematic diagram showing the differentiation of
retinal ganglion cells from human pluripotent stem cells according
to the present invention. With reference to embryological
development of retinal ganglion cells, chemically defined medium,
adhesion of cells in vitro, and differentiation factors were
considered at each stage. Stage and period of embryological
development were planned based on those of mice.
FIG. 4 is a schematic diagram showing the differentiation of
retinal ganglion cells from human pluripotent stem cells according
to the present invention, which was conducted according to a
protocol of treating Wnt3a for 3 days from day 14 to day 17 after
induction of the differentiation (referred to as Protocol A). This
protocol is a prototype of the protocols of the present invention.
The cell type and differentiation markers at respective
differentiation stages are indicated, and photographs of the cells
differentiated according to the protocol are given in FIG.
1A-1F.
FIG. 5 is a schematic diagram showing the differentiation of
retinal ganglion cells from human pluripotent stem cells according
to the present invention, which was conducted according to a
protocol of treating Wnt3a for 3 days from day 11 to day 14 after
induction of the differentiation (referred to as Protocol B).
FIG. 6 is a schematic diagram showing the differentiation of
retinal ganglion cells from human pluripotent stem cells according
to the present invention, which was conducted according to a
protocol of treating Wnt3a for 6 days from day 11 to day 17 after
induction of the differentiation (referred to as Protocol C).
FIG. 7A-7E shows the results of examining cell marker expressions
on day 17 after induction of the differentiation of a human
embryonic stem cell line H9 according to Protocol A.
FIG. 7A shows cells positive to both of the retinal progenitor cell
markers, Rax and Pax6, are observed. Retinal progenitor cells have
the characteristic of being positive to both the markers.
FIG. 7B shows Math5, a marker for immature retinal ganglion cells,
is observed. Compared to the whole cell (DAPI-stained), almost 100%
cells are Math5-positive.
FIG. 7C shows Brn3B, a retinal ganglion cell-specific marker, is
observed in almost all cells. It is indicated that fate of the
cells to retinal ganglion cells is determined by Math5, and then
they begin to differentiate into retinal ganglion cells.
FIG. 7D shows all cells are positive to both of Brn3A (green
staining: nuclear staining) which is one of the retinal ganglion
cell-specific markers, and Tuj1 (red staining: also called
.beta.-tubulin III) which is specific to the cytoplasm of the
neuronal cell.
FIG. 7E shows about 20% of the cells are positive to both of
Islet-1 (red: nuclear staining), which is a marker of the subtype
of retinal ganglion cells, and NF200 (green), which is a neural
axon-specific marker.
*Scale bar: FIG. 7A: 50 .mu.m; FIG. 7B: 200 .mu.m & 50 .mu.m;
FIG. 7C to FIG. 7E: 100 .mu.m & 50 .mu.m.
Nuclear staining of the whole cell: DAPI (blue staining).
FIG. 8 shows the results of flow cytometry to examine marker
expressions on day 39 after induction of the differentiation of a
human embryonic stem cell line H9 according to Protocol A
(experimental results).
FIG. 9A-FIG. 9J shows the results of examining cell marker
expressions on day 39 after induction of the differentiation of a
human embryonic stem cell line H9 according to Protocol A.
FIG. 9A-9C shows the retinal ganglion cell-specific markers, FIG.
9A Pax6, FIG. 9B Brn3B, and FIG. 9C Brn3A observed in FIG. 7A-7E
are still observed.
FIG. 9D shows almost all cells are positive to both of Brn3A
(green: nucleus) and Tuj1 (red: cytoplasmic staining, both axons
and dendrites are stained). The number and thickness of neuronal
axons and dendrites are increased, compared to the cells on day 17
of FIG. 7A-7E. Axonal spiny process and ring, synaptic interaction
between cells, and dendritic arbor and spiny process are
observed.
FIG. 9E shows cells positive to both of Islet-1 (red: nucleus) and
NF200 (green: axon) are observed. The number of axons is increased
and a mature form is observed, compared to the cells on day 17 of
FIG. 7A-FIG. 7E.
FIG. 9F shows Brn3B (green) and Tuj1 (red): The majority of cell
flocs are positive to both Brn3B and Tuj1, which is retinal
ganglion cell-specific staining.
FIG. 9G shows Brn3B (green) and Thy1.2 (red): Nuclear staining for
Brn3B and cytoplasmic staining for Thy1.2 are observed, which are
characteristics of retinal ganglion cells.
FIG. 9H shows Brn3A (green) and Thy1.2 (red): Nuclear staining for
Brn3B and cytoplasmic staining for Thy1.2 are observed, which are
characteristics of retinal ganglion cells.
FIG. 9I shows Map2 (green): Staining of cytoplasm including
dendrites is observed.
FIG. 9J shows results of Western blotting, obtained by reacting
antibodies with the lysate of human embryonic stem cell-derived
retinal ganglion cells on day 39 after induction of
differentiation, indicating retinal ganglion cell-specific
proteins.
*Scale bar: FIG. 9A to FIG. 9C: 50 .mu.m; FIG. 9D: 200 .mu.m, 50
.mu.m & 20 .mu.m; FIG. 9E: 100 .mu.m, 50 .mu.m & 20 .mu.m;
FIG. 9F: 200 .mu.m & 100 .mu.m; FIG. 9G: 20 .mu.m; FIG. 9H: 100
.mu.m; FIG. 9I: 50 .mu.m.
Nuclear staining of the whole cell: DAPI (blue).
FIG. 10A-10H shows the results of examining cell marker expressions
on day 59 after induction of the differentiation of a human
embryonic stem cell line H9 according to Protocol A.
FIG. 10A shows cells positive to both of Brn3A (green: nucleus) and
Tuj1 (red: cytoplasmic staining, both axons and dendrites are
stained) are observed. The number and thickness of axons and
dendrites are increased, compared to the cells on day 39 of FIG.
8.
FIG. 10B shows Tuj1: Axonal spiny process and ring, synaptic
interaction between cells, and dendritic arbor and spiny process
are observed.
FIG. 10C shows NF200: Dendritic spiny process and axon rings are
observed.
FIG. 10D shows Synapsin1: Synapsin1, which is a representative
protein involved in neurotransmitter release, is observed in the
form of functionally mature puncta along dendrites in presynaptic
vesicles, indicating that synapse interaction with other neurons
occurs to transmit neural electric stimuli between cells.
FIG. 10E shows Map2/Vglut1: The majority of presynaptic vesicles
indicated by Synapsin1 of FIG. 10D is glutamatergic vesicles
(Vglut1: red). Presence of Vglut1 along Map2-positive dendrites
(green) is observed. Glutamatergic excitatory neurons are
produced.
FIG. 10F shows Map2/Vgat: The minority of presynaptic vesicles
indicated by Synapsin1 of FIG. 10D is GABAergic vesicles (Vgat:
red). Presence of Vgat along Map2-positive dendrites (green) is
observed.
FIG. 10 G shows PSD-95/Map2: Protein formation is observed in
postsynaptic vesicles, and an excitatory synaptic marker PSD-95 is
distributed along dendrites (Map2).
FIG. 10H shows Synapsin1/PSD-95: physical synapses by super
resolution microscopy: Synapses between presynaptic Synapsin1 and
postsynaptic excitatory PSD-95 are observed. The physical synapses
can be identified by juxtaposition of presynaptic and postsynaptic
vesicle complexes. There are neurotransmitters causing spontaneous
excitatory postsynaptic currents (sEPSCs), which are the
electrophysiological characteristic of "mature retinal ganglion
cells", indicating functional maturation of retinal ganglion
cells.
*Scale bar: FIG. 10A: 100 .mu.m; FIG. 10B: 100 .mu.m & 20
.mu.m; FIG. 10C: 20 .mu.m; FIG. 10D: 50 .mu.m & 20 .mu.m; FIG.
10E: 20 .mu.m; FIG. 10F: 20 .mu.m; FIG. 10G: 50 .mu.m & 1
.mu.m; FIG. 10H: 2 .mu.m & 0.5 .mu.m.
Nuclear staining of cells: DAPI (blue).
FIG. 11A-11I shows the results of examining cell marker expressions
on day 39 after induction of the differentiation of a human
embryonic stem cell line H9 according to Protocol B.
FIG. 11A shows Math5
FIG. 11B shows Brn3B
FIG. 11C shows Brn3A
FIG. 11D shows, FIG. 11E shows Islet-1 (red: nucleus) and NF200
(green: axon)
FIG. 11F shows NF200
FIG. 11G shows Brn3A, Tuj1
FIG. 11H shows Tuj1
FIG. 11I shows Synapsin1/Tuj1
*Scale bar: FIG. 11A to FIG. 11I: 50 .mu.m. Nuclear staining of
FIG. 11H: DAPI (blue).
FIG. 12A-12H shows the results of examining cell marker expressions
on day 39 after induction of the differentiation of a human
embryonic stem cell line H9 according to Protocol C.
FIG. 12A shows Math5
FIG. 12B shows Pax6
FIG. 12C shows Brn3B
FIG. 12D shows Brn3A
FIG. 12E shows Brn3A, Tuj1
FIG. 12E shows Islet-1 (red: nucleus) and NF200 (green: axon)
FIG. 12G shows TrkB: Expression of TrkB, which is a neurotrophin
receptor marker of mature retinal ganglion cells is observed.
FIG. 12H shows Synapsin1/PSD-95
*Scale bar: FIG. 12A to FIG. 12H: 50 .mu.m.
FIG. 13A-FIG. H shows the results of examining cell marker
expressions on day 39 after induction of the differentiation of a
human embryonic stem cell line H7 according to Protocol A.
FIG. 13A shows Math5
FIG. 13B shows Brn3B
FIG. 13C shows Brn3A
Characteristic markers of retinal ganglion cells are observed in
almost all cells.
FIG. 13D shows Brn3A (green: nucleus) and Tuj1 (red: cytoplasmic
staining, both axons and dendrites are stained).
FIG. 13E and FIG. 13F show Islet-1 (red: nucleus) and NF200 (green:
axon)
FIG. 13G shows Synapsin1 (green), Tuj1 (red): A presynaptic marker
Synapsin1 is distributed along Tuj1-positive axons.
FIG. 13H shows Synapsin1/PSD-95: The presynaptic marker Synapsin1
and postsynaptic marker PSD-95 are close to each other, indicating
differentiation of H7 cell line into "mature retinal ganglion
cells" having mature electrophysiological functions by Protocol A,
like H9 cell line.
*Scale bar: FIG. 13A: 50 .mu.m; FIG. 13B and FIG. 13C: 100 .mu.m;
FIG. 13D: 200 .mu.m; FIG. 13E: 200 .mu.m & 20 .mu.m; FIG. 13F:
100 .mu.m; FIG. 13G: 20 .mu.m; FIG. 13H: 20 .mu.m.
Nuclear staining of FIG. 13E: DAPI (blue).
FIG. 14A-FIG. 14L shows the results of examining cell marker
expressions on day 39 after induction of the differentiation of
human induced pluripotent stem cells according to Protocol A.
FIG. 14A shows Math5
FIG. 14B shows Pax6
FIG. 14C shows Brn3B
FIG. 14D shows Brn3A
FIG. 14E shows NF200
FIG. 14F shows Brn3A (red: nucleus) and NF200 (green: axon)
FIG. 14G shows Islet-1 (red: nucleus) and NF200 (green: axon): One
of the subtypes of retinal ganglion cells is observed.
FIG. 14H shows NF200, Islet-1
FIG. 14I shows Brn3A, Tuj1
FIG. 14I shows Tuj1: Differentiation of axons of mature neurons is
observed.
FIG. 14K shows Synapsin1 (green), Tuj1 (red): A presynaptic marker
Synapsin1 is distributed along Tuj1-positive axons.
FIG. 14L shows Synapsin1/PSD-95: The presynaptic marker Synapsin1
and postsynaptic marker PSD-95 are close to each other, indicating
differentiation of human induced pluripotent stem cell line into
"mature retinal ganglion cells" having mature electrophysiological
functions by Protocol A, like H9 cell line.
*Scale bar: FIG. 14A to FIG. 14C, FIG. 14E to FIG. 14H, FIG. 14J,
FIG. 14L: 50 .mu.m; FIG. 14D, FIG. 14I: 100 .mu.m; FIG. 14K: 20
.mu.m.
FIG. 15A-FIG. 15C shows the results of electrophysiological
analysis on day 39 after induction of the differentiation of human
embryonic stem cell line H9 according to Protocol A.
FIG. 15A shows a robust regular-spiking train of action potentials
is observed in response to step current injection (pA) in human
embryonic stem cell-derived retinal ganglion cells, indicating that
the produced retinal ganglion cells acquired mature
electrophysiological characteristics. In general, as neurons
mature, action potentials change from a single short spiking to
multiple lasting spikings in response to current injection.
FIG. 15B shows voltage-gated sodium channel of human embryonic stem
cell-derived retinal ganglion cells: Current responses to step
depolarizations from a holding potential of -80 mV to +40 mV are
superimposed. Fast-activating and inactivating inward sodium
currents were completely blocked by applying tetrodotoxin
(TTX).
FIG. 15C shows spontaneous excitatory postsynaptic currents
(sEPSCs) were detected in human embryonic stem cell-derived retinal
ganglion cells without co-culture with other retinal tissues on day
39 after induction of the differentiation, indicating that
functional excitatory synapses are formed between human embryonic
stem cell-derived retinal ganglion cells.
FIG. 16A-FIG. 16E shows the results of electrophysiological
analysis on day 59 after induction of the differentiation of human
embryonic stem cell line H9 according to Protocol A.
FIG. 16A shows Potassium current: 4-Aminopyridine (4-AP) blocked a
fast-activating fraction of outward potassium current.
FIG. 16B shows a robust regular-spiking train of action potentials
is observed in response to step current injection (pA) in human
embryonic stem cell-derived retinal ganglion cells. The number of
spike was increased, compared to those on day 39 after induction of
the differentiation.
FIG. 16C shows a graph for the number of spikes evoked by step
current injections (pA): The number of spikes increases in
proportion to strength of the current injected.
FIG. 16D shows a staining image of glutamatergic presynaptic
vesicles (Vglut1) distributed in dendrites (Map2-positive).
FIG. 16E shows spontaneous excitatory postsynaptic current
(sEPSCs): An AMPA receptor antagonist CNQX blocked the appearance
of sEPSCs. Glutamatergic excitatory synapses are formed.
FIG. 17A-FIG. 17B shows the results of electrophysiological
analysis on day 96 after induction of the differentiation of human
embryonic stem cell line H9 according to Protocol B.
FIG. 17A shows a spiking train of action potentials is observed in
response to step current injection (pA).
FIG. 17B shows spontaneous excitatory postsynaptic currents
(sEPSCs) are observed. An AMPA receptor antagonist CNQX blocked the
appearance of sEPSCs. Glutamatergic excitatory synapses are
formed.
FIG. 18A-FIG. 18B shows the results of electrophysiological
analysis on day 66 after induction of the differentiation of human
embryonic stem cell line H9 according to Protocol C.
FIG. 18A shows a spiking train of action potentials is observed in
response to step current injection (pA).
FIG. 18B shows spontaneous excitatory postsynaptic currents
(sEPSCs) are observed. Glutamatergic excitatory synapses are
formed.
FIG. 19A-FIG. B shows the results of electrophysiological analysis
of retinal ganglion cells which were matured while supplying a
differentiation medium depleted of IGF-1, Shh, and RA (Retinoic
acid) on day 39 after induction of the differentiation of human
embryonic stem cell line H9 according to Protocol B.
FIG. 19A shows a robust regular-spiking train of action potentials
is observed in response to step current injection (pA) in human
embryonic stem cell-derived retinal ganglion cells on day 96 after
induction of the differentiation.
FIG. 19B shows a spontaneous excitatory postsynaptic currents
(sEPSCs) are observed in human embryonic stem cell-derived retinal
ganglion cells on day 96 after induction of the differentiation.
Glutamatergic excitatory synapses are formed.
FIG. 20A-FIG. 20B shows the results of electrophysiological
analysis on day 39 after induction of the differentiation of human
induced pluripotent stem cells according to Protocol A.
FIG. 20A shows a robust regular-spiking train of action potentials
is observed in response to step current injection (pA) in human
induced pluripotent stem cell-derived retinal ganglion cells,
indicating that the produced retinal ganglion cells acquired mature
electrophysiological characteristics.
FIG. 20B shows Potassium current: 4-Aminopyridine (4-AP) blocked a
fast-activating fraction of outward potassium current.
FIG. 21A-FIG. 21D shows differentiation of human embryonic stem
cell-derived retinal ganglion cells by other Wnt signaling pathway
and Shh receptor activators.
Differentiation was induced according to the time schedule of
Protocol A using 2 .mu.M of a Wnt signaling pathway activator BIO
(6-bromoindirubin-3'-oxime), 50 ng/mL of Norrin, 1 .mu.M of an Shh
receptor activator purmorphamine, and 500 nM of retinoic acid (RA),
in addition to Wnt3a used in the above differentiation method.
Immunofluorescence staining results of the retinal ganglion cell
markers, Islet-1 (red: nucleus) and NF200 (green: axon) in the
cells on day 39 after induction of differentiation are consistent
with the immunofluorescence staining results of Wnt3a and Shh.
*Scale bar: FIG. 21A, FIG. 21C, FIG. 21D: 100 .mu.m; FIG. 21B: 50
.mu.m.
FIG. 22 shows the results of analyzing effects of Wnt3a, Shh and RA
used in the Protocol A differentiation method for production of
retinal ganglion cells. Respective factors were treated according
to a differentiation schedule, and on day 39 after induction of
differentiation, an immunofluorescence assay was performed using
retinal ganglion cell-specific markers.
*Scale bar: 50 .mu.m.
FIG. 23A-FIG. 23B shows cytomorphological microphotographs of human
pluripotent stem cell-derived retinal ganglion cell line after
subculture. The cells are those of passage number 16 after cell
line establishment, that is, being cultured for 3 days from cells
of passage number 15. The subcultured cells of the retinal ganglion
cell line show neuronal morphology in vitro.
FIG. 23A shows a morphology of cells subcultured with Medium 1
FIG. 23B shows a morphology of cells subcultured with Medium 2
Both of the cells subcultured with Medium 1 and Medium 2 show a
distinct neuronal morphology characterized by cytoplasmic
elongation, long neuritis, and phase bright soma.
*left: phase-contrast microscope, 100.times. magnification; right:
200.times. magnification.
FIG. 24A-FIG. 24I shows the results of examining cell marker
expressions of human pluripotent stem cell-derived retinal ganglion
cell line. The cells are those of passage number 17 after cell line
establishment, and immunofluorescence staining was performed at 3
days after passaging the cells of passage number 16. The
characteristic markers of retinal ganglion cells are observed in
almost all cells.
FIG. 24A shows Math5
FIG. 24B shows Brn3B
FIG. 24C shows Brn3A/Tuj1
FIG. 24D shows Synapsin1/Tuj1
FIG. 24E shows NF200
FIG. 24F shows KI67
FIG. 24G shows Thy1.2
FIG. 24H shows NMDAR1
FIG. 24I shows TrkB
*Scale bar: FIG. 24A to FIG. 24I: 50 .mu.m. Nuclear staining: DAPI
(blue).
FIG. 25 shows microscopic imaging of intracellular calcium in human
pluripotent stem cell-derived retinal ganglion cell line,
indicating that the cells have neuronal functions. The human
embryonic stem cell-derived retinal ganglion cells of passage
number 15 were cultured for 3 days. The cells were treated with 1
.mu.M of fluor-4 and stimulated with 1 mM glutamate, and then time
differential images were obtained. As a result, calcium was
detected in many live cells. A serial image spectrum from black
(lowest) to white (highest) represents calcium concentration. In
human embryonic stem cell-derived retinal ganglion cells,
cytoplasmic calcium is considerably increased in response to
glutamate stimulation (head of arrow).
BEST MODE
In order to achieve the above objects, an aspect of the present
invention provides a method of preparing mature retinal ganglion
cells by differentiation of stem cells into mature retinal ganglion
cells, comprising (a) culturing retinal progenitor cells in a
medium containing an IGF1R (insulin-like growth factor-1 receptor)
activator and a Wnt signaling pathway activator to differentiate
them into immature retinal ganglion cells; and (b) culturing the
immature retinal ganglion cells in a medium prepared by removing
the Wnt signaling pathway activator from the medium of step (a) and
adding the IGF1R activator thereto.
Retina is one of the most well-studied organs in the central
nervous system of vertebrates, and detailed morphology, synaptic
connections of neurons, and physiological phenomena of retinal
neurons have been investigated for decades. On the contrary,
structural development of retinal neurons and various functional
mechanisms thereof have not yet been clarified.
Of them, "retinal ganglion cells (RGCs)" are the output neurons
present in the retina of vertebrate, and their axons form the optic
nerve and send an image to the brain. A retinal ganglion cell is a
neuron, that is, a nerve cell as the basic unit of the nervous
system, and has a plurality of dendrites and a single long fibrous
axon. The dendrites form synapses with one another to regulate
electrical activity of neurons and to transmit electrical stimuli
that flow into the cell body. The axon has many branching terminals
containing granules or vesicles which release neurotransmitters in
response to nerve stimulation. A synapse is a point of impulse
transmission between neurons. Chemicals, called neurotransmitters,
pass through a synaptic gap, and nerve impulse is maintained.
During development, retinal ganglion cells are produced from
multipotent retinal progenitor cells. For example, in mice,
development of retinal ganglion cells begins at embryonic day (E)
11.5 and continues until postnatal day (P) 0, and reaches a peak at
E14.5. A major expansion period is known to be between E14.5 and
E17.5, which is variable depending on animals, but is not limited
thereto.
The retinal ganglion cells function to transmit visual information
from various cells of the retina to the optic nerve center of the
brain. Light passed through the eye is converted to electrical
signals in photoreceptor cells, and the electrical signals produced
are transmitted to retinal ganglion cells via various neurons of
the retina. Retinal ganglion cells function to receive these
signals and to transmit them to the optic nerve center of the
brain.
Development of retinal ganglion cells is regulated by a
hierarchical gene regulatory network, and development and
differentiation are mediated by major transcription factors.
The retinal ganglion cells may be divided into immature retinal
ganglion cells and mature retinal ganglion cells according to
differentiation degree.
The "immature retinal ganglion cells" refer to cells that acquire
cytomorphological characteristics and protein and genetic marker
characteristics of retinal ganglion cells (Barres, et al., Neuron.
1988; 1: 791-803.). The "mature retinal ganglion cells" refer to
cells that are in the mature state as neurons after acquiring the
above characteristics of the "immature retinal ganglion cells".
That is, they have the morphological characteristics of axonal
growth, arbor, and ring; dendritic arbor, stratification, and ring;
dendritic spiny process; synaptic bouton; etc., and the protein and
genetic marker characteristics of presynaptic or postsynaptic
protein morphology and various receptor characteristics as
excitatory nerves. The electrophysiological characteristics of the
"mature retinal ganglion cells" are formation of spontaneous
excitatory postsynaptic currents (sEPSCs), indicating functional
maturation capable of forming synaptic connections between retinal
ganglion cells (Pfrieger Barres, Science. 1997; 277: 1684-7.).
The "immature retinal ganglion cells" refer to cells at an early
stage of differentiation from retinal progenitor cells into retinal
ganglion cells. These immature retinal ganglion cells are cells
that are fated to be retinal ganglion cells from retinal progenitor
cells or cells that are fated to be retinal ganglion cells, but
lack morphological and physiological characteristics of mature
retinal ganglion cells. In the present invention, the immature
retinal ganglion cells and early retinal ganglion cells are used
interchangeably.
The "mature retinal ganglion cells" refer to retinal ganglion cells
at a late stage of differentiation from retinal progenitor cells
into retinal ganglion cells or retinal ganglion cells which have
already differentiated. The mature retinal ganglion cells have a
physiological characteristic of producing a high-frequency action
potential and a morphological characteristic of having long axons,
but are not limited thereto.
Whether retinal ganglion cells are immature retinal ganglion cells
or mature retinal ganglion cells can be determined by identifying
the expression levels of markers specific to respective cells, and
morphological and physiological characteristics thereof (e.g.,
electrochemical characteristics).
First, transcription factors playing an important role in
development and differentiation of retinal ganglion cells are as
follows: transcription factors involved in the development of
retinal ganglion cells are ATOH7 (Math5), Brn3 family (Brn3A,
Brn3B, and Brn3C), and Isl1 (Islet-1).
ATOH7, also called Math5, is a factor involved in determining the
fate of retinal progenitor cells to retinal ganglion cells, and is
essential for formation of retinal ganglion cells. Therefore, Math5
is preferably expressed in the immature retinal ganglion cells.
Further, POU4F2 and POU4F1 (also called Brn3B and Brn3A,
respectively) and Isl1 (Islet-1) downstream of ATOH7 in the gene
network are known not to be required for the birth of RGC, but are
known to be required for RGC differentiation and survival. They are
known to be expressed at an early stage of differentiation of
retinal ganglion cells and also expressed in mature retinal
ganglion cells.
In detail, the Brn3 family is involved in regulation of RGC
differentiation, dendritic stratification of RGCs, and axonal
projection of RGCs during development. Of the Brn3 family, Brn3B is
a factor expressed early during RGC development, and is one of the
earliest RGC markers, and acts as an important factor in axon
growth and survival of RGCs.
In mice, expression of Brn3A begins at embryonic day (E) 12.5, and
in rats, Brn3A is expressed in 92.2% of the RGC population. Brn3B
is known to act in axon formation of RGCs, whereas Brn3A is known
to be involved in dendrite formation.
Isl1 is a homeodomain LIM protein expressed during retina
development. Isl1 gene is activated immediately after the birth of
RGCs, and its expression is identical with that of POU4F2 before
the embryonic day (E) 14.5. Further, Isl1 is a factor required for
RGC differentiation and survival, and its expression is governed by
ATOH7. Isl1 expression is initiated at mouse embryonic day (E) 11.5
in completely divided cells, and Isl1 is known to be co-expressed
with Brn3B in some of Brn3B-positive RGC cells.
The above transcription factors play an important role in
generation of retinal ganglion cells, and thus immature retinal
ganglion cells may express (1) Math5 and/or (2) one, specifically
two, and more specifically three selected from the group consisting
of Brn3B, Brn3A and Islet1. Further, expressions of these marker
genes may be increased, compared to those in retinal progenitor
cells, but are not limited thereto.
Further, the immature retinal ganglion cells may be those
expressing NF200 or/and Tuj1. NF200 is a protein called
neurofilament 200 kDa, and expressed at an early stage of
differentiation of retinal ganglion cells. Tuj1 is also known to be
expressed at an early stage of differentiation of retinal ganglion
cells. Therefore, these two genes may be used to distinguish
retinal ganglion cells, specifically, between immature retinal
ganglion cells and mature retinal ganglion cells, but are not
limited thereto. Additionally, marker genes specific to mature
retinal ganglion cells are Thy1.2, TrkB, NMDAR1, Map2, Vglut1,
PSD-95, synaptophysin, and synapsin1. These genes are marker genes
known to be expressed in mature retinal ganglion cells.
Therefore, mature retinal ganglion cells may express one,
specifically two, and more specifically 4, 5, 6, 7, 8, 9 and 10
marker genes selected from the group consisting of Brn3B, Brn3A,
Islet-1, NF200, Tuj1, Thy1.2, TrkB, NMDAR1, Map2, Vglut1, PSD-95,
synaptophysin and synapsin1, but are not limited thereto. Further,
mature retinal ganglion cells may show high expressions of these
genes, compared to retinal progenitor cells or immature retinal
ganglion cells, but are not limited thereto.
The method of the present invention is a method capable of
differentiating retinal progenitor cells into mature retinal
ganglion cells via immature retinal ganglion cells in a high yield,
characterized by culturing retinal progenitor cells in a medium
containing a Wnt signaling pathway activator to differentiate them
into immature retinal ganglion cells, and then culturing the
immature retinal ganglion cells in a medium without the Wnt
signaling pathway activator to differentiate them into mature
retinal ganglion cells. That is, technical features of the present
invention are to determine the fate of stem cells to retinal
ganglion cells by treatment of stem cells at a specific
differentiation stage with the Wnt signaling pathway activator and
to differentiate 60% to 95% or more of various retinal cell
populations into mature retinal ganglion cells by removing the Wnt
signaling pathway activator. This method of inducing highly
efficient differentiation into mature retinal ganglion cells was
developed by the present invention for the first time.
A detailed description of the method of the present invention will
be given below.
In the method of the present invention, step (a) is a step of
culturing retinal progenitor cells in a medium containing an IGF1R
activator and a Wnt signaling pathway activator to differentiate
them into immature retinal ganglion cells. Step (a) may be
performed for 1 day to 10 days, but is not limited thereto.
As used herein, the term "retinal progenitor cell" refers to a
multipotent progenitor cell which can differentiate into cells
present in the retina or retinal pigmented epithelial cells. The
retinal progenitor cells are characterized by expressing one, two,
or three or more markers selected from the group consisting of Rax,
Pax6, Chx10, Otx2, Sox2, Lhx2, Six3, Six6 and Mitf, and in
particular, by expressing one or two of Rax and Pax6, but are not
limited thereto.
In connection with retinal development, as mentioned above, retinal
progenitor cells are able to differentiate into various types of
intraretinal cells (rod and cone photoreceptor cells, retinal
ganglion cell, horizontal cells, bipolar cells, amacrine cells,
Muller glial cells, etc.) and retinal pigmented epithelium,
featuring positive expression of markers such as Crx, recoverin,
rhodopsin, red/green opsin, blue opsin, peripherin2, PDE6B, SAG,
Islet1/NF200, Prox1, PKC-a, Hu C/D, GFAP, ZO-1, and RPE65. However,
the expression level and positive rate of these markers become
weaker in retinal progenitor cells than in mature retinal cells or
retinal pigmented epithelium, but are not limited thereto.
A medium for differentiation of retinal progenitor cells into
immature retinal ganglion cells is characterized by containing all
of the IGF1R activator and Wnt signaling pathway activator.
Further, the medium for differentiation of retinal progenitor cells
into immature retinal ganglion cells may contain, but is not
limited to, a BMP (bone morphogenetic protein) signaling pathway
inhibitor and an FGF (fibroblast growth factor) signaling pathway
activator, in addition to the IGF1R activator and Wnt signaling
pathway activator. Further, the medium may be a DMEM/F12 medium
containing 1% B27 supplement and 1% N2 supplement in addition to
the above-described materials, but is not limited thereto.
As used herein, the term "IGF1R activator" refers to a substance
which activates IGF-1 (insulin-like growth factor-1) receptor
(IGF1R), a member of the tyrosine kinase receptor family. Activated
IGF1R interacts with insulin receptor substrates (IRS). In turn,
IRS activated by IGF1R acts as an activator of one pathway
consisting of PI3K, Akt, and mTOR, and the other pathway consisting
of Raf, MEK, and ERK (Ryan & Goss, Oncologist. 2008; 13:
16-24). IGF-1 and IGF-2 fall within the range of the IGF1R
activator. IGF-1, having a molecular structure similar to insulin,
is implicated in cell growth, cell proliferation, differentiation,
and cell death, but is not limited thereto.
Any IGF1R activator may be used without limitation in the present
invention, as long as it activates IGF1R. It is exemplified by
IGF-1 or IGF-2, and specifically, IGF-1, but is not limited
thereto.
The medium used for differentiation of retinal progenitor cells
into immature retinal ganglion cells contains the IGF1R activator
in an amount of specifically 0.01 ng/mL to 100 ng/mL, more
specifically 0.1 ng/mL to 50 ng/mL, much more specifically 1 ng/mL
to 20 ng/mL, and most specifically 10 ng/mL, but is not limited
thereto.
As used herein, the term "Wnt signaling pathway activator" refers
to a substance activating the Wnt signaling pathway which is known
to regulate various processes during embryogenesis, including cell
fate determination, reconstruction of organization, polarity,
morphology, adhesion and growth, and the maintenance and
proliferation of undifferentiated cells. Any activator may be
included within the Wnt signaling pathway without limitation, as
long as it transduces Wnt-mediated or .beta.-catenin-mediated
signals. The Wnt signaling pathway is a series of processes that
are initiated by the binding of a first trigger of the Wnt
signaling pathway, Wnt, to its receptor or mediated by the
stabilization of .beta.-catenin, which is a downstream target in
the intracellular Wnt signaling pathway. The Wnt signaling pathway
activator is, but is not particularly limited to, as follows.
1) By directly adding a Wnt protein: Wnt, a first trigger of the
Wnt signaling pathway, is a family of secreted glycoproteins. 19
Wnts have been identified: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4,
Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b,
Wnt10a, Wnt10b, Wnt11, and Wnt16b.
2) By increasing the level of .beta.-catenin: Most cells respond to
Wnt signaling pathway by an increase in the level of
.beta.-catenin. That is, an increase in dephosphorylated
.beta.-catenin level (or the stabilization) means the translocation
of .beta.-catenin into the nucleus.
3) By phosphorylation of dishevelled or phosphorylation of a
Wnt-associated receptor, LRP tail.
4) By using GSK3 (glycogen synthase kinase 3) inhibitors: Lithium
(Li), LiCl, bivalent Zn, BIO (6-bromoindirubin-3'-oxime), SB216763,
SB415286, QS11 hydrate, TWS119, Kenpaullone, alsterpaullone,
indirubin-3'-oxime, TDZD-8, and Ro 31-8220 methanesulfonate
salt.
5) By blocking negative regulators of the Wnt signaling pathway,
such as Axin and APC, or by using RNAi.
6) With activators of the Wnt pathway, such as norrin and
R-spondin2: Norrin binds to Frizzled4 receptor while R-spondin2
interacts with Frizzled8 and LRP6.
7) By gene transfer, including transfection: Either Wnt
overexpression constructs or .beta.-catenin overexpression
constructs may be used.
In the present invention, the Wnt signaling pathway activators may
be employed without limitation as long as it is able to activate
the Wnt signaling pathway. They are specifically Wnt1, Wnt2, Wnt2b,
Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b,
Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16b; substances increasing
.beta.-catenin levels; GSK3 inhibitors such as lithium, LiCl,
bivalent zinc, BIO, SB216763, SB415286, CHIR99021, QS11 hydrate,
TWS119, Kenpaullone, alsterpaullone, indirubin-3'-oxime, TDZD-8 or
Ro 31-8220 methanesulfonate salt; Axin inhibitors, APC inhibitors,
norrin or R-spondin 2, and more specifically, Wnt3a, Wnt1, Wnt5a,
Wnt11, Norrin, LiCl, BIO or SB415286, but are not particularly
limited thereto.
The Wnt signaling pathway activator used for inducing
differentiation of retinal progenitor cells into immature retinal
ganglion cells may be used in an amount of 0.01 ng/mL to 500 ng/mL,
specifically in an amount of 0.1 ng/mL to 200 ng/mL, and more
specifically in an amount of 1 ng/mL to 100 ng/mL, except for LiCl,
BIO and SB415286. Among the Wnt signaling pathway activators, the
content of LiCl in the medium is 0.1 mM to 50 mM, specifically 0.5
mM to 10 mM, and more specifically 1 mM to 10 mM; the content of
BIO in the medium is 0.1 .mu.M to 50 .mu.M, specifically 0.1 .mu.M
to 10 .mu.M, and more specifically 0.5 .mu.M to 5 .mu.M; the
content of SB415286 in the medium is 0.1 .mu.M to 500 .mu.M,
specifically 1 .mu.M to 100 .mu.M, and more specifically 5 .mu.M to
50 .mu.M.
As used herein, the term "BMP signaling pathway inhibitor" means a
group of substances capable of inhibiting BMP signaling pathway.
BMPs belong to a signaling pathway protein belonging to the
TGF-.beta. (transforming growth factor-.beta.) superfamily and are
involved in early-prenatal differentiation, prenatal tissue
formation, and homeostasis of adult tissues. Extracellular secreted
BMPs bind to Type I and Type II serine/threonine kinase receptors,
initiating the BMP signaling pathway. A Type II receptor
phosphorylates a Type I receptor. The phosphorylated Type I
receptor then phosphorylates the intracellular substrate Smad
protein, mediating the intracellular signal transduction pathway.
Smad proteins regulated by receptors are called R-Smads (Receptor
regulated Smad), and Smad-1, 2, 3, 5 and 8 belong to R-Smads. They
can bind the intracellular common partner Smad (Co-Smad) Smad-4.
They migrate into and accumulate in the nucleus where they act as
transcription factors and participate in the regulation of target
gene expression (Yamamoto & Oelgeschlager, Naturwissenschaften.
2004; 91: 519-34). A BMP signaling pathway inhibitor refers to a
substance which blocks the binding of extracellular BMP to
cell-surface receptors. Examples of BMP signal pathway inhibitors
include Noggin, chordin, twisted gastrulation (Tsg), cerberus,
coco, gremlin, PRDC (protein related to DAN and Cerberus), DAN
(differential screening-selected gene aberrative in neuroblastoma),
dante, follistatin, USAG-1 (uterine sensitization-associated gene
1), dorsomorphin and sclerostin. By inhibiting BMP signal
transduction, Noggin plays a key role in neural induction and in
ventralizing dorsal neuroectoderm or mesoderm. Also, Noggin binds
to the BMPs, BMP-2, BMP-4 and BMP-7, and blocks these BMPs from
binding to their receptors (Yanagita, Cytokine Growth Factor Rev.
2005; 16: 309-17).
In the present invention, any BMP signaling pathway inhibitor may
be employed, as long as it is able to inhibit BMP signal
transduction. Examples thereof may include Noggin, chordin, twisted
gastrulation, cerberus, coco, gremlin, PRDC, DAN, dante,
follistatin, USAG-1 (uterine sensitization-associated gene 1),
dorsomorphin and sclerostin, but are not limited thereto. In an
embodiment of the present invention, Noggin was used.
In the present invention, the content of the BMP signaling pathway
inhibitor in the medium for inducing differentiation of the retinal
progenitor cells into immature retinal ganglion cells is 0.01 ng/mL
to 100 ng/mL, specifically 0.1 ng/mL to 50 ng/mL, more specifically
0.5 ng/mL to 20 ng/mL, and most specifically 10 ng/mL, but is not
limited thereto.
As used herein, the term "FGF signaling pathway activator" refers
to a substance capable of inducing or promoting FGF signal
transduction. FGF is a factor involved in mitogenesis including
cell proliferation and cell differentiation, angiogenesis, bone
morphogenesis and neural induction. Twenty-two members of the FGF
family have been identified so far. There are four members of the
FGF receptor family (FGFR). Their mRNAs bind to alternatively
spliced receptor variants to activate the FGF receptors. Activated
FGFR mediates the signal through a Ras/Raf/MeK pathway in the
cytoplasm to activate MAP kinase, which in turn induces signal
transduction (Bottcher & Niehrs, Endocr Rev. 2005; 26: 63-77).
Of the FGF family, FGF2, also known as basic FGF (bFGF), binds
mainly to FGFR 1b, FGFR 1c, FGFR 2c, FGFR 3c, and FGFR 44, and
strongly activates FGFR 1c and FGFR 3c. Activators of FGFR 1c and
FGFR 3c as well as FGF1, FGF4, FGF8, FGF9, FGF17, and FGF19 may be
used as substitutes, but are not limited thereto.
In the present invention, any FGF signaling pathway activator may
be used without limitation, as long as it is able to stimulate FGF
signal transduction. Examples thereof may include an FGFR 1c or
FGFR 3c activator, FGF1, FGF2, FGF4, FGF8, FGF9, FGF17 or FGF19,
but are not limited thereto. In an embodiment of the present
invention, FGF2 was used.
In the present invention, the content of the FGF signaling pathway
activator in the medium for inducing differentiation of the retinal
progenitor cells into immature retinal ganglion cells is 0.01 ng/mL
to 100 ng/mL, specifically 0.1 ng/mL to 50 ng/mL, more specifically
1 ng/mL to 20 ng/mL, and most specifically 5 ng/mL, but is not
particularly limited thereto.
Further, the retinal progenitor cells of step (a) may be obtained
by (a') culturing stem cells in a medium containing an IGF1R
activator, a Wnt signaling pathway inhibitor, and a BMP signaling
pathway inhibitor to differentiate them into eye field precursors
in the form of floating aggregates; and (b') culturing the eye
field precursors of step (a') in the form of floating aggregates in
a medium prepared by supplementing the medium of step (a') with an
FGF signaling pathway activator to differentiate them into retinal
progenitor cells.
Any stem cells may be used without particular limitation, as long
as they are able to differentiate into retinal progenitor cells,
and the stem cells may be selected from the group consisting of
bone marrow stem cells (BMS), cord blood stem cells, amniotic fluid
stem cells, fat stem cells, retinal stem cells (RSCs), intraretinal
Muller glial cells, embryonic stem cells (ESCs), induced pluipotent
stem cells (iPSCs), and somatic cell nuclear transfer cells
(SCNTCs).
These stem cells may be differentiated into eye field precursors by
culturing them in a medium containing an IGF1R activator, a Wnt
signaling pathway inhibitor, and a BMP signaling pathway inhibitor.
In this regard, the IGF1R activator, the BMP signaling pathway
inhibitor, and concentrations thereof are the same as in the above
medium used for inducing differentiation into immature retinal
ganglion cells
As used herein, the term "Wnt signaling pathway inhibitor" refers
to a factor which interrupts interaction between the extracellular
Wnt protein and the membrane protein Frizzled receptor or a
co-receptor LRP, or inhibits intracellular Wnt-mediated signal
transduction. In the present invention, any Wnt signaling pathway
inhibitor may be used without particular limitation, as long as it
inhibits Wnt-mediated signal transduction. Examples of the Wnt
signaling pathway inhibitors may include the Dkk (Dickkopf) family
(Dkk-1, Dkk-2, Dkk-3 and Dkk-4), which are antagonists of the
co-receptor LRP, Wise, Wnt antagonists (sFRP: secreted
Frizzled-related protein) family, a Frizzled-CRD domain, WIF-1 (Wnt
inhibitory factor-1), IWP-2, IWP-3, IWP-4, cerberus, Wnt
antibodies, dominant negative Wnt proteins, overexpression of Axin,
overexpression of GSK (glycogen synthase kinase), dominant negative
TCF, dominant negative dishevelled or casein kinase inhibitors
(CKI-7, D4476 etc.), but are not limited thereto. In an embodiment
of the present invention, Dkk-1 was used.
Further, Wnt signal transduction may be inhibited by suppressing
each component involved in the Wnt pathway, such as RNAi, in
addition to the Wnt signaling pathway inhibitor.
In the medium, the content of the Wnt signaling pathway inhibitor
used for differentiating the stem cells into retinal progenitor
cells via eye field precursors may be 0.01 ng/mL to 10,000 ng/mL,
but is not particularly limited thereto.
As used herein, the term "eye field precursor" refers to a cell
expressing specific markers (eye field transcription factors;
Zuber, et al., Development, 2003; 130: 5155-67) found in a
progenitor for the eye field of the forebrain neural plate during
embryonic development. The eye field precursors are characterized
by expressing one, two, or three or more markers selected from the
group consisting of Six3, Rax, Pax6, Otx2, Lhx2 and Six6, but are
not limited thereto.
The eye field precursors have a morphology of floating aggregates.
In this regard, the floating aggregate refers to a cell mass
floating in a medium which is generated when a floc of embryonic
stem cells is cultured for at least one day in a non-adhesive plate
without feeding mouse embryonic fibroblasts (MEF) and sera.
Depending on the composition of the medium supplied, the eye field
precursors may express eye field transcription factors.
Further, step (a') may be performed for 1 day to 30 days, and step
(b') may be performed for 5 days to 15 days, but are not limited
thereto.
In the present invention, the floating aggregates of eye field
precursors of step (b') may be grown adhering to a plate which is
coated with an extracellular matrix selected from the group
consisting of poly-D-lysine, laminin, poly-L-lysine, matrigel,
agar, polyornithine, gelatin, collagen, fibronectin and
vitronectin.
The cell population per floating aggregate which adheres to the
plate is the number of cells which is the most highly efficient. A
floating aggregate of eye field precursors may consist of 200 cells
to 400 cells, but is not limited thereto.
Further, the medium of step (a') may be a DMEM/F12 medium further
containing 10% knockout serum replacement and 1% B27 supplement, in
addition to an IGF1R activator, a Wnt signaling pathway inhibitor,
and a BMP signaling pathway inhibitor.
Meanwhile, the medium of step (b') may be a DMEM/F12 medium further
containing 1% B27 supplement and 1% N2 supplement, in addition to
the above-described IGF1R activator, Wnt signaling pathway
inhibitor, BMP signaling pathway inhibitor, and FGF signaling
pathway activator.
In the method of the present invention, step (b) is a step of
culturing immature retinal ganglion cells which are differentiated
in step (a). Immature retinal ganglion cells may be differentiated
into mature retinal ganglion cells by step (b).
For highly efficient differentiation into mature retinal ganglion
cells, the medium used in step (b) is characterized by containing
no Wnt signaling pathway activator which is contained in the medium
of step (a).
More specifically, step (b) may be a step of culturing the cells of
step (a) in a medium prepared by removing the BMP signaling pathway
inhibitor, the FGF signaling pathway activator, and the Wnt
signaling pathway activator from the medium of step (a), and more
specifically, in a medium prepared by removing the BMP signaling
pathway inhibitor, the FGF signaling pathway activator, and the Wnt
signaling pathway activator from the medium of step (a) and adding
an Shh (sonic hedgehog) signaling pathway activator thereto.
Further, step (b) may comprise (i) culturing immature retinal
ganglion cells in a medium containing the IGF1R activator, the BMP
signaling pathway inhibitor, and the FGF signaling pathway
activator by removing the Wnt signaling pathway activator from the
medium of step (a); and (ii) culturing immature retinal ganglion
cells in a medium prepared by removing the BMP signaling pathway
inhibitor and the FGF signaling pathway activator from the medium
of step (i) and adding an Shh (sonic hedgehog) signaling pathway
activator thereto.
Meanwhile, as used herein, the term "removing" may be interpreted
as using a medium that does not contain a subject to be removed.
Also, the term "a medium whose composition does not comprise X"
means all of the medium that does not contain X.
Further, the medium may be a DMEM/F12 medium containing 1% B27
supplement and 1% N2 supplement, in addition to the above-described
substances, but is not limited thereto.
As used herein, the term "Shh (sonic hedgehog) signaling pathway
activator" refers to a substance that induces or activates the Shh
signal transduction. Shh is known as a signal transducer associated
with the regulation of various processes during embryogenesis,
including cell fate determination, polarity, morphology,
proliferation, and differentiation (Bertrand & Dahmane, Trends
Cell Biol. 2006; 16: 597-605). The Shh signaling pathway involves
two transmembrane proteins, Ptc (Patched) and Smo (Smoothened). In
the absence of Shh, Ptc interacts with and inhibits Smo. On the
contrary, in the presence of shh, Shh binds to Ptc, and Smo is no
longer inhibited, leading to Ci/Gli protein in the cytoplasm
entering the nucleus and acting as a transcriptional activator for
target genes. No particular limitations are imparted to the Shh
signaling pathway activator if it is able to enhance the
Shh-mediated signaling pathway. Examples of the Shh signaling
pathway activators may include proteins belonging to the hedgehog
family (e.g., Shh), inhibitors of inhibitory function of Ptc on
Smo, Smo receptor activators, Shh receptor activators (e.g. Hg--Ag,
purmorphamine, etc.), substances increasing Ci/Gli family levels,
inhibitors of the intracellular degradation of Ci/Gli proteins, and
Shh overexpression constructs or Ci/Gli overexpression constructs
resulting from transfection.
In the present invention, any Shh signaling pathway activator may
be used, as long as it is able to activate the Shh signaling
pathway. Examples thereof may include Shh, Smo receptor activators,
inhibitors of inhibitory function of Ptc on Smo, substances
increasing Ci/Gli family levels, inhibitors of the intracellular
degradation of Ci/Gli factors, and an Shh receptor activator,
Hg--Ag or purmorphamine, but are not limited thereto. More specific
examples of the Shh signaling pathway activator may include Shh and
purmorphamine.
In the present invention, the content of the Shh signaling pathway
activator in the medium used for differentiating immature retinal
ganglion cells into mature retinal ganglion cells is 0.1 ng/mL to
5,000 ng/mL, specifically 1 ng/mL to 2,500 ng/mL, more specifically
10 ng/mL to 1,000 ng/mL, and most specifically 250 ng/mL, but is
not particularly limited thereto.
Step (b) may be performed for 1 day to 200 days.
For highly efficient differentiation into mature retinal ganglion
cells, the method may further include the step of (c) culturing the
cells cultured in step (b) in a medium prepared by adding RA
(retinoic acid) to the medium of step (b).
As used herein, the term "RA (retinoic acid)" refers to a
metabolite of vitamin A, which is a lipophilic molecule. There are
two types of RA: all-trans retinoic acid and 9-cis retinoic acid.
RA is translocated into the nucleus where it binds to RARs
(retinoic acid receptors) and RXR (retinoid X receptors)
respectively and participates in the regulation of target gene
transcription, but is not limited thereto.
RA used in the method of the present invention may be
trans-retinoic acid and cis-retinoic acid, and the concentration of
RA to be used may be 0.5 nM to 10,000 nM, specifically 5 nM to
5,000 nM, more specifically 50 nM to 2,000 nM, and much more
specifically 500 nM, but is not particularly limited thereto.
Step (c) may be performed for 1 day to 120 days, but is not limited
thereto.
The method of the present invention may further include the step of
(d) culturing immature retinal ganglion cells or mature retinal
ganglion cells in a medium prepared by removing one or more, two or
more, or three selected from the group consisting of an IGF1R
activator, Shh signaling pathway activator, and RA, more
specifically, by removing all of the IGF1R activator, Shh signaling
pathway activator and RA from the medium of step (c).
According to embodiments of the present invention, although
immature retinal ganglion cells undergoing maturation were cultured
in a medium free of all of the IGF1R activator, Shh signaling
pathway activator, and RA, they showed maturity equivalent to those
cultured in a medium containing all of the components.
Further, the method of the present invention may include the step
of determining whether the desired cells after the above-described
step, for example, immature retinal ganglion cells after step (a),
are differentiated or not, or determining whether mature retinal
ganglion cells after step (b), (c) or (d) are differentiated or
not.
Differentiation of the desired cells, specifically, immature
retinal ganglion cells and mature retinal ganglion cells, may be
determined by measuring expressions of marker genes specific to the
cells, and/or by identifying morphological characteristics and/or
physiological characteristics.
The measurement of the marker gene expression may be performed by
measuring the expression level of mRNA or protein of the marker
gene, and measurement of the expression level may be performed by
using various methods known in the art.
Any technique of analyzing the marker gene expression at an mRNA
level may be used in the present invention without limitations, as
long as it is a method of analyzing mRNA well-known in the art.
Examples thereof may include reverse transcriptase polymerase chain
reaction, competitive reverse transcriptase polymerase chain
reaction, real-time polymerase chain reaction, Rnase Protection
Assay, Northern blotting, and DNA chip assay.
Further, any technique of analyzing the marker gene expression at a
protein level may be used in the present invention without
limitations, as long as it is a method of analyzing a protein
well-known in the art. Examples thereof may include Western
blotting, ELISA, radioimmunoassay, radioimmunodiffusion,
Ouchterlony immunodiffusion, rocket immunoelectrophoresis,
immunohistostaining, immunoprecipitation assay, complement fixation
assay, FACS, and protein chip assay.
Compared to the pre-differentiative retinal progenitor cells, the
mature retinal ganglion cells differentiated according to the
present invention exhibit one or more of the following features:
(1) an increased expression level of Brn3B; (2) an increased
expression level of Brn3A; (3) an increased expression level of
Islet1; (4) an increased expression level of NF200; (5) an
increased expression level of Tul1; (6) an increased expression
level of Thy1.2; (7) an increased expression level of TrkB; (8) an
increased expression level of NMDAR1; (9) an increased expression
level of Map2; (10) an increased expression level of Vglut1; (11)
an increased expression level of PSD-95; (12) an increased
expression level of Synaptophysin; and (13) an increased expression
level of Synapsin1.
An increase or decrease in the expression levels of the genes may
be identified using antibodies against the proteins encoded by the
genes or using methods well-known to those skilled in the art, such
as RT-PCR. As they show more of the features, the differentiated
cells are defined as being closer to the mature retinal ganglion
cells. Preferably, the mature retinal ganglion cells show one or
more, specifically 2 or more, more specifically 3 or more, much
more specifically 4 or more, and most specifically 5 or more of the
features. Preferably, more than approximately 40%, 60%, 80%, 90%,
95% or 98% of the population of the cells after differentiation
according to the method of the present invention have the desired
features. Higher ratios are more preferable, but are not limited
thereto.
Another aspect of the present invention provides a method of
differentiating immature retinal ganglion cells into mature retinal
ganglion cells, including the step of culturing the immature
retinal ganglion cells in a medium containing the IGF1R activator
and the Shh signaling pathway activator.
The immature retinal ganglion cells, IGF1R activator, Shh signaling
pathway activator, and mature retinal ganglion cells are the same
as described above.
The medium which is not applied to the method of differentiating
immature retinal ganglion cells into mature retinal ganglion cells
is characterized by containing no Wnt signaling pathway
activator.
In the method, the IGF1R activator may be used specifically in an
amount of 0.01 ng/mL to 100 ng/mL, more specifically in an amount
of 0.1 ng/mL to 50 ng/mL, much more specifically in an amount of 1
ng/mL to 20 ng/mL, and most specifically in an amount of 10 ng/mL,
but is not particularly limited thereto.
In the method, the Shh signaling pathway activator may be used in
an amount of 0.1 ng/mL to 5,000 ng/mL, specifically in an amount of
1 ng/mL to 2,500 ng/mL, more specifically in an amount of 10 ng/mL
to 1,000 ng/mL, and most specifically in an amount of 250 ng/mL,
but is not particularly limited thereto.
Further, the medium may be a DMEM/F12 medium containing 1% B27
supplement and 1% N2 supplement in addition to the above-described
substances, but is not limited thereto.
The method may further include the step of culturing immature
retinal ganglion cells in a medium prepared by adding RA (retinoic
acid) to the medium containing the IGF1R activator and the Shh
signaling pathway activator. Herein, "adding" may be interpreted as
replacing the previous medium with a fresh medium containing the
above substance together with the previous medium composition, as
well as adding the substance to the medium.
Herein, RA is the same as described above, and the concentration of
RA to be used may be 0.5 nM to 10,000 nM, specifically 5 nM to
5,000 nM, more specifically 50 nM to 2,000 nM, and much more
specifically 500 nM, but is not particularly limited thereto.
The method may further include the step of culturing immature
retinal ganglion cells or mature retinal ganglion cells in a medium
prepared by removing one or more selected from the group consisting
of an IGF1R activator, Shh signaling pathway activator, and RA, and
more specifically, all of the IGF1R activator, Shh signaling
pathway activator, and RA from the above-described medium.
Still another aspect of the present invention provides immature
retinal ganglion cells and mature retinal ganglion cells prepared
according to the above-described method of the present
invention.
The method, the immature retinal ganglion cells, and the mature
retinal ganglion cells are the same as described above.
Still another aspect of the present invention provides a method of
screening for a death inhibitor or a proliferation promoter of
mature retinal ganglion cells, comprising (a) treating the mature
retinal ganglion cells obtained and isolated by the above method of
differentiating into mature retinal ganglion cells with a death
inhibitor candidate or a proliferation promoter candidate of mature
retinal ganglion cells; and (b) determining that the candidate is a
death inhibitor or a proliferation promoter of mature retinal
ganglion cells when the candidate inhibits death of the mature
retinal ganglion cells or promotes proliferation of the mature
retinal ganglion cells, compared to a non-candidate-treated
group.
The method of differentiating into mature retinal ganglion, and the
mature retinal ganglion cells are the same as described above.
Step (a) is a step of treating the mature retinal ganglion cells
obtained and isolated by the above method of differentiating into
mature retinal ganglion cells according to the present invention
with a death inhibitor candidate or a proliferation promoter
candidate of mature retinal ganglion cells.
The mature retinal ganglion cells to be treated with the candidate
may be, but is not particularly limited to, mature retinal ganglion
cells which are differentiated by applying the method of the
present invention to stem cells derived from a healthy person or a
patient.
As used herein, the term "death inhibitor of mature retinal
ganglion cells" refers to a substance capable of inhibiting death
of mature retinal ganglion cells. Specifically, the death inhibitor
may include a substance capable of reducing death of mature retinal
ganglion cells, to which death induction conditions of mature
retinal ganglion cells are applied, compared to those to which the
death induction conditions are not applied. The type of the death
inhibitor is not particularly limited, and the death inhibitor
includes compounds, proteins, peptides, and nucleic acids.
As used herein, the term "proliferation promoter of mature retinal
ganglion cells" refers to a substance capable of inducing or
promoting proliferation of mature retinal ganglion cells.
Specifically, the proliferation promoter may include a substance
capable of increasing proliferation of mature retinal ganglion
cells, to which the proliferation promoter is applied, compared to
those to which the proliferation promoter is not applied. The type
of the proliferation promoter is not particularly limited, and the
proliferation promoter includes compounds, proteins, peptides, and
nucleic acids.
The method of the present invention may include the step of (b)
determining that the candidate is a death inhibitor or a
proliferation promoter of mature retinal ganglion cells when the
candidate inhibits death of the mature retinal ganglion cells or
promotes proliferation of the mature retinal ganglion cells,
compared to a non-candidate-treated group. Further, the death
inhibitor or proliferation promoter of mature retinal ganglion
cells may be a therapeutic agent for glaucoma or optic
neuropathy.
As used herein, the term "glaucoma" refers to a disease having
optic nerve damage accompanied by loss of retinal ganglion
cells.
As used herein, the term "optic neuropathy" refers to a disease
resulting from visual disorders caused by gradual loss of the
retinal ganglion cells and their axons which constitute the retina.
The optic neuropathy includes optic neuritis, ischemic optic
neuropathy, toxic or deficiency optic neuropathy, hereditary optic
neuropathy, and optic atrophy.
As described above, glaucoma and optic neuropathy are diseases
accompanied by loss of retinal ganglion cells, and thus a substance
capable of inhibiting death of retinal ganglion cells or promoting
proliferation thereof may be used as a therapeutic agent for
glaucoma or optic neuropathy.
Still another aspect of the present invention provides a kit of
screening for the death inhibitor or the proliferation promoter of
mature retinal ganglion cells, including the mature retinal
ganglion cells prepared by the above-described method of the
present invention. Further, the kit may be used for screening of
the therapeutic agent for glaucoma or optic neuropathy.
The mature retinal ganglion cells, the death inhibitor, and the
proliferation promoter are the same as described above.
The kit may include various tools and/or reagents capable of
screening the death inhibitor or proliferation promoter of retinal
ganglion cells in addition to mature retinal ganglion cells, which
are known in the art. If necessary, the kit may further include a
tube for mixing each component, a well plate, and an instruction
manual describing use thereof.
Experimental procedures, reagents, and reaction conditions that may
be used in the methods may be those commonly known in the art and
will be obvious to those skilled in the art.
Still another aspect of the present invention provides a
pharmaceutical composition for treating glaucoma or optic
neuropathy, including the mature retinal ganglion cells prepared by
the above-described method of the present invention.
The mature retinal ganglion cells, glaucoma and optic neuropathy
are the same as described above.
For use in the therapeutic composition of the present invention,
differentiation of mature retinal ganglion cells from stem cells,
for example, human embryonic stem cells or induced pluripotent stem
cells, is induced in vitro, mature retinal ganglion cells are
proliferated and differentiated in a large amount and then
administered to a patient having the above disease. The therapeutic
composition may be formulated into dosage forms general in the art,
for example, injectable formulations. The composition may be
directly transplanted into a retinal site using a surgical
procedure or may be intravenously injected and migrate to a retinal
site. The therapeutic composition of the present invention may
further include an immunosuppressant to suppress immune rejection
responses to grafts. The composition may further include a
pharmaceutically acceptable carrier. The administration dose of the
therapeutic composition of the present invention may vary depending
on the severity of the patient, the route, method, and frequency of
administration, the time period of treatment, the patient's age,
sex, and disease severity, and may be easily determined by those
skilled in the art according to various factors well-known in the
medical art.
Still another aspect of the present invention provides a method of
treating glaucoma or optic neuropathy, including the step of
administering the mature retinal ganglion cells prepared by the
method of the present invention to a subject suspected of having
glaucoma or optic neuropathy.
The method, mature retinal ganglion cells, glaucoma and optic
neuropathy are the same as described above.
The mature retinal ganglion cells of the present invention may be
administered to any animal, and the animal may include livestock,
such as cows, pigs, sheep, horses, dogs, mice, rats, cats, etc., as
well as humans and primates.
As used herein, the term "administration" refers to the
introduction of the composition of the present invention into a
subject suspected of having glaucoma or optic neuropathy by a
suitable route, including the transplantation of the differentiated
cells. Any administration route by which the composition of the
present invention reaches a tissue of interest may be employed in
the present invention. Intraretinal injection is preferred.
Still another aspect of the present invention provides a method of
preparing a mature retinal ganglion cell line, comprising (a)
culturing retinal progenitor cells in a medium containing the IGF1R
(insulin-like growth factor-1 receptor) activator and the Wnt
signaling pathway activator to differentiate them into immature
retinal ganglion cells; and (b) culturing the immature retinal
ganglion cells in a medium prepared by removing the Wnt signaling
pathway activator from the medium of step (a) and adding the IGF1R
activator thereto.
The medium may further include the Shh signaling pathway activator
or RA (retinoic acid), or both.
Further, the method may further comprise separating the cells that
are cultured in the medium containing the IGF1R activator and the
Shh signaling pathway activator, and then culturing the cells in
(i) a medium containing L-glutamine, mercaptoethanol, and
insulin/transferrin/selenium-X, or (ii) a medium containing
L-glutamine, mercaptoethanol, FGF2, IGF-1 and EGF. The medium may
specifically be an IMDM medium containing L-glutamine,
mercaptoethanol, and insulin/transferrin/selenium-X, or an IMDM
medium containing L-glutamine, mercaptoethanol, FGF2, IGF-1 and
EGF. In an embodiment of the present invention, Medium 1 containing
IMDM, 15% FBS, 1 mM L-glutamine, 0.1 mM mercaptoethanol, and 1%
insulin/transferrin/selenium-X, and Medium 2 containing IMDM, 15%
FBS, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 5 ng/mL of FGF2, 10
ng/mL of IGF-1, and 5 ng/mL of human recombinant EGF were used.
MODE FOR INVENTION
Hereinafter, the present invention will be described in detail with
reference to Examples. However, these Examples are for illustrative
purposes only, and the invention is not intended to be limited by
these Examples.
EXAMPLE 1
Culture of Stem Cells
<1-1> Culture of Human Embryonic Stem Cells
Human embryonic stem cell (hESC) lines H9 (WA09, normal karyotype
XX) and H7 (WA07, normal karyotype, XX) were purchased from the
WiCell Research Institute (Madison, Wis.).
The human embryonic stem cells were allowed to proliferate
undifferentiated (H9 cells: passages 26 to 41; H7 cells: passages
23 to 32) by culturing them over feeder cells, such as irradiated
mouse embryonic fibroblasts (MEF, Global Stem, Gaithersburg, Md.)
or mitomycin-treated mouse embryonic fibroblasts (EmbryoMax Primary
Mouse Embryo Fibroblasts, Millipore, Billerica, Mass.) in the
following medium: embryonic stem cell medium [DMEM/F12 (Invitrogen,
Grand Island, N.Y.) liquid, 20% (v/v) KnockOut serum replacement
(Invitrogen, Carlsbad, Calif.), 1 mM L-glutamine (Invitrogen), 0.1
mM nonessential amino acids (Invitrogen), 0.1 mM mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo.), 4 ng/mL human recombinant FGF2
(human recombinant basic fibroblast growth factor,
Invitrogen)].
While the medium was replaced every day, the undifferentiated stem
cells (FIG. 1A) were passaged at a ratio of 1:15 to 1:18 every six
or seven days manually or with collagenase V (collagenase IV,
Invitrogen), and then transferred onto fresh mouse embryonic
fibroblast feeder cells. During the passage of the human embryonic
stem cells, immunochemical staining with OCT-4 and SSEA-4
(Chemicon, Temecula, Calif.), which are antigens specific to
undifferentiated human embryonic stem cells, was conducted at
regular intervals of time to monitor the degree of differentiation.
Cells that were found to have undergone differentiation were
removed. Meanwhile, the presence of mycoplasma contamination in the
embryonic stem cell line, which could have an undesirable effect on
the differentiation of human embryonic stem cells, was regularly
monitored using a MycoAlert mycoplasma detection kit (Lonza,
Rockland, Me.).
<1-2> Generation and Culture of Human Induced Pluripotent
Stem Cell (iPSC)
Human induced pluripotent stem cells were prepared by transfecting
stem cell genes (SOX2, KLF4, OCT4, L-MYC, LIN28, and small hairpin
RNA for p53) into BJ1 fibroblasts (neonatal foreskin fibroblast,
ATCC) using episomal plasmid vectors (Okita, Nat method 2011; 8:
409). The human induced pluripotent stem cells were allowed to
proliferate undifferentiated (passages 5 to 9) by culturing them
over feeder cells, such as irradiated mouse embryonic fibroblasts
(MEF, Global Stem, Gaithersburg, Md., USA) or mitomycin-treated
mouse SNL cells (SNL 76/7 cell line, ECACC, Porton Down, UK) in the
following medium: embryonic stem cell medium [DMEM/F12 (Invitrogen,
Grand Island, N.Y., USA) liquid, 20% (v/v) KnockOut serum
replacement (Invitrogen, Carlsbad, Calif., USA), 1 mM L-glutamine
(Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM
mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), and 4 ng/mL human
recombinant FGF2 (Invitrogen)].
While the medium was replaced every day, the undifferentiated stem
cells (FIG. 2A) were passaged at a ratio of 1:12 to 1:15 every six
or seven days manually or with collagenase IV (Invitrogen), and
then transferred onto fresh mouse embryonic fibroblast feeder
cells. During the passage of the human induced pluripotent stem
cells, immunochemical staining (FIGS. 2B and 2C) with alkaline
phosphatase (Sigma-Aldrich), NANOG (Abcam, Cambridge, Mass.) and
SSEA-4 (Chemicon, Temecula, Calif.), which are antigens specific to
undifferentiated human induced pluripotent stem cells, was
conducted at regular intervals of time to monitor the degree of
differentiation. Cells that were identified to have undergone
differentiation were removed. To evaluate pluripotency of the
undifferentiated human induced pluripotent stem cells, a teratoma
assay was performed. 10 weeks after transplantation of
1.times.10.sup.7 of human induced pluripotent stem cells into the
dorsal surface of an immunosuppressed model, rd/SCID mouse,
teratomas thus formed were removed, followed by H&E staining
(FIG. 2D).
The presence of mycoplasma contamination in the human induced
pluripotent stem cells, which could have an undesirable effect on
the differentiation of human induced pluripotent stem cells, was
regularly monitored using a MycoAlert mycoplasma detection kit
(Lonza, Rockland, Me.).
EXAMPLE 2
Differentiation from Human Embryonic Stem Cells or Human Induced
Pluripotent Stem Cells into Eye Field Precursors
First, human embryonic stem cells or human induced pluripotent stem
cells cultured by the method of Example 1 were separated from the
mouse embryonic fibroblasts and transferred onto 6-well ultra-low
attachment plates (Corning Incorporated, Corning, N.Y., USA),
respectively.
To the human embryonic stem cells or human induced pluripotent stem
cells in the 6-well ultra-low attachment plates was added a medium
for inducing differentiation into eye field precursors [DMEM/F12,
10% KnockOut serum replacement, 1 mM L-glutamine, 0.1 mM
nonessential amino acids, 0.1 mM mercaptoethanol, 1% B27 supplement
(Invitrogen), 1 ng/mL recombinant Noggin (R&D Systems), 1 ng/mL
recombinant Dkk-1 (recombinant Dickkopf-1, R&D Systems), and 5
ng/mL recombinant IGF-1 (recombinant insulin-like growth factor-1,
R&D Systems)]. While the medium was aspirated at 3 days and
then replaced with a fresh medium, the cells were cultured for 4
days to 5 days to generate eye field precursors in the form of
floating aggregates in the medium (FIGS. 1B, 3).
EXAMPLE 3
Differentiation from Eye Field Precursors into Retinal Progenitor
Cells
The eye field precursors (floating aggregates) generated in Example
2 were seeded at a density of 53.+-.8 cells per well (292.+-.53
cells/floating aggregate) into 6-well poly-D-lysine/laminin-coated
plates (BD Biosciences), at a density of 30.+-.5 cells per well on
12-well poly-D-lysine/laminin-coated plates, and at a density of
12.+-.4 cells per well on 8-well poly-D-lysine/laminin-coated
plates, and then cultured for 10 days to generate retinal
progenitor cells (FIG. 1C), with the supply of a medium for
inducing differentiation into retinal progenitor cells [DMEM/F12
(Invitrogen), 1 mM L-glutamine (Invitrogen), 0.1 mM nonessential
amino acids (Invitrogen), 0.1 mM mercaptoethanol (Sigma-Aldrich),
1% B27 supplement, 1% N2 supplement (Invitrogen), 10 ng/mL Dkk-1,
10 ng/mL Noggin, 10 ng/mL IGF-1, and 5 ng/mL FGF2] (Protocol A)
(FIGS. 3 and 4). Protocol B (FIG. 5) and C (FIG. 6) were performed
by supplying the present medium for 7 days.
EXAMPLE 4
Differentiation from Retinal Progenitor Cells into Immature Retinal
Ganglion Cells (RGCs)
The retinal progenitor cells generated in Example 3 were cultured
in a medium for inducing differentiation into immature retinal
ganglion cells [DMEM/F12(Invitrogen), 1 mM L-glutamine
(Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM
mercaptoethanol (Sigma-Aldrich), 1% B27 supplement (Invitrogen), 1%
N2 supplement (Invitrogen), 10 ng/mL Noggin, 10 ng/mL IGF-1, 5
ng/mL FGF2, 50 ng/mL recombinant Wnt3a (recombinant Wnt3a, R&D
Systems)] for 3 days, thus giving immature retinal ganglion cells
(FIGS. 1D, 3 to 5). Protocol C was performed by supplying the
present medium for 6 days (FIG. 6).
EXAMPLE 5
Differentiation from Immature Retinal Ganglion Cells into Mature
Retinal Ganglion Cells: Step 1
The immature retinal ganglion cells generated in Example 4 were
induced to differentiate into mature retinal ganglion cells by
supplying a differentiation medium [DMEM/F12 (Invitrogen), 1 mM
L-glutamine (Invitrogen), 0.1 mM nonessential amino acids
(Invitrogen), 0.1 mM mercaptoethanol (Sigma-Aldrich), 1% B27
supplement (Invitrogen), 1% N2 supplement (Invitrogen), 10 ng/mL
IGF-1, 250 ng/mL recombinant Shh (recombinant Sonic Hedgehog amino
terminal peptide, Shh, R&D Systems)] for 5 days (FIG. 1E, 3 to
6).
EXAMPLE 6
Differentiation from Immature Retinal Ganglion Cells into Mature
Retinal Ganglion Cells: Step 2
The retinal ganglion cells generated in Example 5 were matured by
supplying a differentiation medium [DMEM/F12 (Invitrogen), 1 mM
L-glutamine (Invitrogen), 0.1 mM nonessential amino acids
(Invitrogen), 0.1 mM mercaptoethanol (Sigma-Aldrich), 1% B27
supplement (Invitrogen), 1% N2 supplement (Invitrogen), 10 ng/mL
IGF-1, 250 ng/mL Shh, 500 nM all-trans retinoic acid (RA,
Sigma-Aldrich)] for 17 days (FIG. 1E, 3 to 6). All the media of
Examples 2 to 6 were replaced with fresh ones every two or three
days, and the cells were cultured at 37.degree. C. in a 5% CO.sub.2
atmosphere (FIG. 1F, 3 to 6). The present medium for retinal
ganglion cells was used to culture the cells until day 120 of
differentiation.
EXAMPLE 7
Assay for Cellular Differentiation-Related Markers
<7-1> Immunochemical Staining and Identification of Cellular
Differentiation-Related Marker Protein Expression
The differentiation of the cells obtained in Examples 3 to 6 was
examined using an immunochemical staining method as follows.
The eye field precursors (floating aggregates) were cultured in
8-well poly-D-lysine/laminin-coated slides (BD Biosciences,
Bedford, Mass.) under the same conditions that were used for
differentiation into the retinal progenitor cells, the immature
retinal ganglion cells, and the mature retinal ganglion cells. The
cells completely cultured in each step were fixed with 4%
paraformaldehyde (Sigma-Aldrich), after which non-specific
reactions were blocked with PBS containing 3% BSA (Jackson
Immunoresearch Laboratory, Bar Harbor, Me., USA) and 0.25% Triton
X-100 (Sigma-Aldrich).
After being blocked for 90 min, the slides in each differentiation
step were incubated overnight at 4.degree. C. with antibodies
specific to cells of each differentiation step as given in Table 1.
Before use, these antibodies were diluted in a PBS solution
containing 1% BSA and 0.25% Triton X-100. The cells cultured on the
slides in each step were washed three times for 5 min with PBS and
incubated at room temperature for 2 hr with species-specific
secondary antibody conjugated with Cy3 (1:800, Jackson
Immunoresearch Laboratory) or Alexa488 (1:500, Invitrogen) (Table
1). A standard material suitable for the primary and the secondary
antibodies was used to examine non-specific staining or interaction
between the antibodies. Thereafter, the cells were washed three
times for 5 min with PBS, counterstained with DAPI
(4',6-diamidino-2 phenylindole) and mounted in Vectashield (Vector
Laboratories), followed by visualization under an epifluorescence
microscope (Nikon Eclipse, E800, Tokyo, Japan) and a confocal
microscope (Zeiss LSM510, Carl Zeiss, Inc, Thornwood, N.Y., USA).
500 cells were counted from 20 microscopic fields randomly selected
at 400 magnification and evaluated for positive responses to each
antibody. Positive responses to antibodies were determined after at
least three evaluations.
<7-2> Flow Cytometry and Identification of Cellular
Differentiation-Related Marker Protein Expression
Cells were detached by 0.05% trypsin (Invitrogen) on days 17 and 39
after induction of differentiation. Cells were fixed with a
Cytofix/Cytoperm buffer solution (Biosciences), and washed with a
Perm/Wash buffer solution (BD Biosciences). Cells were reacted with
primary antibody at 4.degree. C. for 30 min, and secondary antibody
at 4.degree. C. for 20 min (Table 1). The reaction samples were
analyzed by FACSCalibur (BD Biosciences) and FlowJo software
(Treestar).
<7-3> Identification of Retinal Ganglion Cell-Specific Marker
by Western Blotting
The cultured cells were lysed on day 39 after induction of
differentiation with a protein extraction buffer solution
containing a protease inhibitor. Total protein concentration was
measured using a Bradford protein assay kit. An equal amount of the
protein was loaded on polyacrylamide gel (Any kD Mini-PROTEAN TGX
precast polyacrylamide gels, Bio-Rad, Hercules, Calif.), and then
transferred onto a polyvinylidene difluoride membrane (Milipore,
Billerica, Mass.). This membrane was reacted with primary antibody
(Table 1, Brn3B 1:1,000, Brn3A 1:1,000, Tuj1 1:1000, NF200 1:1,000,
Actin 1:2,000) and secondary antibody (HRP-conjugated secondary
antibody, Goat anti-mouse or anti-rabbit IgG, diluted 1:2,000,
Santa Cruz, Calif.), and then the reacted proteins were detected
using chemiluminescence detection kit (Amersham ECL, GE Healthcare
Life technology, Piscataway, N.J.).
TABLE-US-00001 TABLE 1 Antibodies used in immunofluorescence assay
Dilution for Catalogue Antibody Species ICF* Supplier No. Brn3A
Rabbit 1:500 gift from Eric N/A Turner Brn3B Rabbit 1:500 gift from
Eric N/A Turner Islet-1 Mouse 1:10 DSHB 40.2D6 KI67 Mouse 1:200
Vector vp-k452 Laboratories Map2 Mouse 1:1,000 Sigma-Aldrich M1406
Map2 Rabbit 1:500 Milipore AB5622 Math5 Rabbit 1:500 Abcam ab78046
Nanog Rabbit 1:500 Abcam ab80892 Neurofilament- Rabbit 1:1,000
Sigma-Aldrich N4142 200 NMDAR1 Mouse 1:250 BD Bio- 556308 sciences
Pax6 Mouse 1:1 DSHB PAX6 PSD-95 Mouse 1:500 NeuroMab 75-028 Rax
Rabbit 1:250 Abcam ab23340 SSEA4 Mouse 1:100 Chemicon MAB4304
Synapsin1 Rabbit 1:500 Stressgen VAP- SV060E Thy-1.2 Mouse 1:250
eBioscience 14-0903-81 TrkB Mouse 1:100 Santa Cruz sc-136991
.beta.-tubulin Mouse 1:1,000 Covance MMS-435P (Tuj1) Vgat Rabbit
1:1,000 Synaptic 131 003 Systems Vglut1 Rabbit 1:1,000 Synaptic 135
303 Systems Anti-mouse Alexa Goat 1:500 Molecular A11029 Fluor 488
Probes Anti-rabbit Alexa Goat 1:500 Molecular A11034 Fluor 488
Probes Anti-mouse Cy3 Goat 1:800 Jackson 115-165- Laboratory 062
Anti-rabbit Cy3 Goat 1:800 Jackson 111-165- Laboratory 045
Anti-mouse R- Goat 1:800 Jackson 115-116- Phycoerythrin Laboratory
146 Anti-rabbit R- Goat 1:800 Jackson 115-116- Phycoerythrin
Laboratory 144 *ICF: Immunocytofluorescence staining
As a result of identifying differentiation-related marker
expression, flow cytometry results (Table 2A) of markers of cells
obtained by Protocol A of Example 4 on day 17 after induction of
differentiation showed that retinal progenitor cell markers, Rax-
and Pax6-positive cells were 95.8.+-.1.4% and 93.8.+-.0.7%,
respectively (FIG. 7A). Meanwhile, retinal ganglion cell-specific
markers, Math5, Brn3B- and Brn3A-positive cells were 95.2.+-.1.2%,
96.8.+-.0.6%, and 97.5.+-.0.9%, respectively. In mice, Math5, which
is expressed at an early stage of retinal ganglion cells, is known
to be detected from embryonic day (E) 11 to 1 day after birth, and
Brn3B and Brn3A are known to be detected at a time of retinal
ganglion cell generation during embryonic development and continue
for their life. Further, these markers are co-expressed in most
retinal ganglion cells. Islet-1, which is a marker of the subtype
of retinal ganglion cells, was detected as 16.4.+-.2.7%. On day 17
after induction of differentiation, the immunofluorescence staining
results and positive rates are consistent with the results of flow
cytometry. In nuclei of most cells, Math5 and Brn3B were strongly
stained (FIGS. 7B and 7C). The cytoplasm of Brn3A (nuclear
staining)-positive cells are positive to neuron-specific marker
Tuj1 (98.2.+-.0.4%) (Table 2A, FIG. 7D), indicating their
differentiation into retinal ganglion cells, which are a type of
neuron. About 1/5 of the cells positive to NF200 which is expressed
in axons of retinal ganglion cells were positive to Islet-1, which
is a marker of the subtype of retinal ganglion cells (Table 2A,
FIG. 7E). 1/3of the total retinal ganglion cells are reported to be
Islet-1-positive retinal ganglion cells. Maturation characteristics
of axonal and dendritic arbor, ring, and spiny process were not
observed yet.
Flow cytometry results (Table 2B, FIG. 8) of markers of cells
obtained by Protocol A of Example 6 on day 39 after induction of
differentiation showed that Pax6-positive cells were 82.3.+-.3.5%.
Pax6 is known to be expressed in retinal progenitor cells and also
to continue to be expressed after differentiation into retinal
ganglion cells (FIG. 9A). Math5-, Brn3B (FIGS. 9B, 9F, 9G)-, and
Brn3A (FIGS. 9C, 9D, 9H)-positive cells were 77.3.+-.2.7%,
84.7.+-.0.8%, 89.9.+-.2.3%, respectively. It seems that Math5 was
reduced with cell differentiation. Islet-1-positive cells were
16.4.+-.2.7% on day 17 after induction of differentiation and
increased to 32.8.+-.7.1% on day 39 after induction of
differentiation, indicating that 1/3of the total retinal ganglion
cells are Islet-1-positive (FIG. 9E). Meanwhile, cell proliferation
marker KI67-positive cells were remarkably reduced from
81.0.+-.3.5% to 18.4.+-.3.5% on day 17 after induction of
differentiation. The immunofluorescence staining results showed
that axonal ring and spiny process, and dendritic arbor, ring, and
spiny process were observed in retinal ganglion cells, indicating
maturation (FIGS. 9F to 9I). This maturation further progressed on
day 59 after induction of differentiation (FIG. 10).
Results of Western blot analysis also showed strong expressions of
retinal ganglion cell-specific markers, Brn3B and Brn3A and
neuron-specific markers, NF200 and Tuj1, as in the results of
immunochemical staining and flow cytometry (FIG. 9J).
TABLE-US-00002 TABLE 2 Flow cytometry results of H9-derived RGC on
days 17 and 39 after differentiation according to Protocol A A) day
17 B) day 39 Marker Mean, %(n = 3) SEM Marker Mean, %(n = 3) SEM
Brn3A 97.5 0.9 Brn3A 89.9 2.3 Brn3B 96.8 0.6 Brn3B 84.7 0.8 Islet-1
16.4 2.7 Islet-1 32.8 7.1 KI67 81.0 3.5 KI67 18.4 3.5 Math5 95.2
1.2 Math5 77.3 2.7 NF200 98.3 0.4 NF200 96.5 0.9 Pax6 93.8 0.7 Pax6
82.3 3.5 Tuj1 98.2 0.4 Tuj1 94.7 2.5 Rax 95.8 1.4
On day 39 after induction of differentiation into retinal ganglion
cells according to Protocol B (FIG. 5) and Protocol C (FIG. 6) of
Examples 5 and 6, marker protein expressions were identified. The
results of flow cytometry of cell markers (Tables 3A and 3B) showed
that cells positive to retinal ganglion cell-specific markers,
Math5, Brn3B and Brn3A and neuronal axon and dendrite markers, Tuj1
and NF200 were similar to those of the prototype Protocol A, but
the subtype marker Islet-1-positive cells were reduced to
12.4.+-.2.5% and 7.8.+-.1.6% in Protocols B and C. On the other
hand, cytomorphology was examined by immunofluorescence staining.
As a result, cells differentiated according to Protocol B showed
positive rates of axons and dendrites and maturity being similar to
those of the cells according to Protocol A, but low positive rates
of presynaptic and postsynaptic vesicles having electrical
functions, compared to Protocol A (FIG. 11). Cells differentiated
according to Protocol C showed active generation of presynaptic and
postsynaptic vesicles, but relatively low maturity of axon and
dendrites (FIG. 12).
TABLE-US-00003 TABLE 3 Flow cytometry results of H9-derived RGC on
day 39 after differentiation according to Protocols B and C A)
Protocol B B) Protocol C Marker Mean, %(n = 3) SEM Marker Mean, %(n
= 3) SEM Brn3A 89.8 5.0 Brn3A 91.6 1.0 Brn3B 83.3 1.5 Brn3B 86.0
2.4 Islet-1 12.4 2.5 Islet-1 7.8 1.6 KI67 15.0 1.0 KI67 15.9 4.7
Math5 93.6 1.1 Math5 91.0 1.9 NF200 89.6 3.8 NF200 94.1 1.7 Pax6
80.1 4.7 Pax6 75.7 3.9 Tuj1 94.1 1.8 Tuj1 95.6 0.5
Human embryonic stem cell line H7, other than H9, and human induced
pluripotent stem cells were differentiated into retinal ganglion
cells according to Protocol A of Example 6. On day 39 after
induction of differentiation, immunofluorescence staining of the
cell markers and flow cytometry were performed. As a result,
percentages of Math5-, Brn3B- and Brn3A-positive human embryonic
stem cell H7 were 95.0%, 90.2% and 94.4%, respectively, which are
the same as in H9 cell line (Table 4A). Immunofluorescence staining
results showed mature features of axons and dendrites and active
generation of presynaptic and postsynaptic vesicles having
electrical functions (FIG. 13).
Percentages of Math5-, Brn3B- and Brn3A-positive human induced
pluripotent stem cell were 73.0%, 61.4% and 77.0%, respectively,
which are relatively lower than in H9 cell line (Table 4B).
Immunofluorescence staining results showed mature features of axons
and dendrites and active generation of presynaptic and postsynaptic
vesicles having electrical functions (FIG. 14).
TABLE-US-00004 TABLE 4 Flow cytometry results of H7 and
iPSC-derived RGC on day 39 after differentiation according to
Protocol A A) H7-derived RGCs B) iPSC-derived RGCs Marker Mean, %(n
= 2) Marker Mean, %(n = 2) Brn3A 94.4 Brn3A 77.0 Brn3B 90.2 Brn3B
61.4 Islet-1 17.8 Islet-1 12.0 KI67 29.1 KI67 25.4 Math5 95.0 Math5
73.0 NF200 95.5 NF200 87.5 Pax6 87.8 Pax6 56.2 Tuj1 90.5 Tuj1
89.0
EXAMPLE 8
Test of Electrophysiological Properties and Functions of Retinal
Ganglion Cells Differentiated from Human Pluripotent Stem Cells
Whole-cell voltage recordings and current-clamp recordings were
performed at 32.+-.1.degree. C. by infusing a recording chamber
with artificial cerebrospinal fluid at a rate of 1 mL/min to 1.5
mL/min. The artificial cerebrospinal fluid was composed of 125 mM
NaCl, 25 mM NaHCO.sub.3, 2.5 mM KCl, 1.25 mM NaH.sub.2PO.sub.4, 2
mM CaCl.sub.2, 1 mM MgCl.sub.2, 20 mM glucose, 1.2 mM pyruvate and
0.4 mM Na-ascorbate, and saturated at pH 7.4 with 95% O.sub.2 and
5% CO.sub.2. A patch pipette having a tip resistance of 3.5
M.OMEGA. to 4.5 M.OMEGA. was used. A series resistance after
whole-cell configuration was between 10 M.OMEGA. and 15 M.OMEGA..
Recordings were performed using an EPC-10 amplifier (HEKA,
Lambrecht-Pfalz, Germany), and upon recording, a pipette solution
containing 143 mM K-gluconate, 7 mM KCl, 15 mM HEPES, 4 mM MgATP,
0.3 mM NaGTP, 4 mM Na-ascorbate and 0.1 mM EGTA was used, and pH
was adjusted to 7.3 using KOH. Spontaneous excitatory postsynaptic
currents (sEPSCs) were recorded at a holding potential of -70 mV.
To identify the patterns of spontaneous postsynaptic current,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 .mu.M),
D-(-)-2-amino-5-phosphonopentanoic acid (AP5, 50 .mu.M) and
(+)-bicuculline (50 .mu.M) were injected to a bath solution of the
experimental chamber (Sigma-Aldrich). CNQX blocks AMPA receptor,
AP5 blocks NMDA receptor, and bicuculline blocks GABA receptor.
Na.sup.+ currents were blocked by tetrodotoxin (TTX, 1 .mu.M), and
K.sup.+ currents were blocked by 4-aminopyridine (4-AP, 1 mM)
(Sigma-Aldrich).
<8-1> Test of Electrophysiological Properties of Retinal
Ganglion Cells Differentiated from Human Pluripotent Stem Cells
To test electrical properties of the human pluripotent stem
cell-derived retinal ganglion cells differentiated according to the
present invention, retinal ganglion cells differentiated according
to Protocol A was first evaluated. In the evaluation of action
potentials on day 39 after induction of differentiation, a robust
regular-spiking train of action potentials was observed in response
to step current injection (FIG. 15A). The results of whole-cell
patch clamp recording showed the presence of voltage-gated sodium
channel, which was blocked by tetrodotoxin (FIG. 15B). Therefore,
it was confirmed that human pluripotent stem cell-derived retinal
ganglion cells were differentiated into electrically excitable
cells. In the evaluation of action potentials on day 59 after
induction of differentiation, action potentials were found to be
more mature in response to step current injection than those on day
39 after induction of differentiation (FIG. 16B). It was reported
that during embryological development, neurons develop into mature
firing cells over days. An identical process is found in vivo
(Connors et al. 1982; McCormick & Prince, 1987). Similar
results are also observed in retinal ganglion cells differentiated
according to Protocols B (FIG. 17A) and C (FIG. 18A) and human
induced pluripotent stem cells (FIG. 20). The retinal ganglion
cells differentiated according to the protocol of the present
invention showed increased electrical maturation and function over
time.
<8-2> Test of Electrophysiological Functions of Retinal
Ganglion Cells Differentiated from Human Pluripotent Stem Cells
Retinal ganglion cells differentiated from human pluripotent stem
cells according to the present invention form functional excitatory
synapses. Synaptogenesis is a critical step in the development of
neural networks. The formation of physical synapses among human
pluripotent stem cell-derived retinal ganglion cells was detected
using super-resolution microscopy to visualize presynaptic and
postsynaptic protein localization. Synapses were defined as regions
of hundreds of nanometers in diameter found near dendrites detected
by MAP2 staining where proteins specific to the pre- and
postsynaptic compartments were juxtaposed. Antibodies against
presynaptic and postsynaptic proteins were used to identify
synapses: Antibodies against the excitatory glutamatergic
postsynaptic protein, PSD-95 (glutamatergic postsynaptic density
95) and the presynaptic protein, synapsin1 were used. Foci of
PSD-95 generated from pluripotent stem cell-derived retinal
ganglion cells were abundant along dendrites or in the 100 nm size
range thereof, and juxtaposed, but non-overlapping, with
presynaptic vesicle, synapsin1 (FIGS. 10G and 10H). These are also
found in retinal ganglion cells derived from another human
embryonic stem cell line H7 (FIGS. 13G and 13H) and human induced
pluripotent stem cells (FIGS. 14K and 14L), indicating formation of
physical synapses among human pluripotent stem cell-derived retinal
ganglion cells.
Meanwhile, physical synapses formed among the cells were
electrophysiologically evaluated. Spontaneous excitatory
postsynaptic currents (sEPSCs) were detected without co-culture
with other retinal tissues on day 39 after induction of
differentiation from human embryonic stem cell-derived retinal
ganglion cells (FIG. 15C), indicating formation of functional
excitatory synapses among human embryonic stem cell-derived retinal
ganglion cells. Further, frequency of sEPSCs and depth of spiking
were increased when evaluated on day 59 after induction of
differentiation (FIG. 16E), implying that neuronal maturation
progressed over time. The sEPSCs were found to be blocked by CNQX,
suggesting that they are one of AMPA-mediated excitatory
glutamatergic neurons. Similar results are also observed in the
retinal ganglion cells differentiated according to Protocols B
(FIG. 17B) and C (FIG. 18B).
EXAMPLE 9
Comparison Between Protocols for Differentiation from Human
Embryonic Stem Cells or Human Induced Pluripotent Stem Cells into
Retinal Photoreceptor Cells and Retinal Ganglion Cells
The present inventors disclosed a method of differentiating retinal
cells from stem cells in the previous patents (Korean Patent NO.
10-1268741 (2013 May 22) and WO2011/043591 (2011 Apr. 14)).
According to the methods disclosed in the above documents,
differentiation of photoreceptor cells from retinal progenitor
cells was maximized to about 80%. However, their differentiation
into retinal ganglion cells was partially progressed to only about
6%.
<9-1> Differentiation from Human Embryonic Stem Cells or
Human Induced Pluripotent Stem Cells into Retinal Photoreceptor
Cells
Differentiation from human embryonic stem cells or human induced
plur